Small molecule stimulators of neuronal growth

ABSTRACT

Provided herein are small molecule stimulators of neuronal growth, their preparation, and their use for treatment of neurological disorders. In one embodiment, provided herein are methods of treatment, prevention, or amelioration of a variety of medical conditions associated with neurological disorders using the compounds and compositions provided herein.

RELATED APPLICATION DATA

This application claims the benefit of and is a Continuation Applicationof U.S. application Ser. No. 11/140,618, filed May 26, 2005 which claimsthe benefit of U.S. Provisional Application No. 60/574,433, filed May26, 2004; each of which is hereby incorporated by reference in itsentirety.

GRANT INFORMATION

This invention was made with government support under Grant No. RGY 0072sponsored by Human Frontiers Science Program. The United Statesgovernment has certain rights in this invention.

FIELD

Provided herein are small molecule stimulators of neuronal growth, theirpreparation, and their use for treatment of neurological disorders. Inanother embodiment, provided herein are methods of treatment,prevention, or amelioration of a variety of medical conditionsassociated with neurological disorders using the compounds andcompositions provided herein.

BACKGROUND

Neurological disorders afflict large numbers of people in the world. Indisorders ranging from neurodegenerative diseases (e.g., Alzheimer's andParkinson's diseases) to traumatic spinal cord injuries, there is a needfor molecules that promote neuronal growth. While some proteins areknown that can stimulate neuronal growth, few small molecules are knownthat can stimulate neuronal growth.

Chondroitin sulfate (CS) glycosaminoglycans are sulfated polysaccharidesimplicated in cell division, neuronal development, and spinal cordinjury (Mizuguchi, S.; Uyama, T.; Kitagawa, H.; Nomura, K. H.; Dejima,K.; Gengyo-Ando, K.; Nitani, S.; Sugahara, K.; Nomura, K. Nature 2003,423, 443-448; Sugahara, K.; Mikami, T.; Uyama, T.; Mizuguchi, S.;Nomura, K.; Kitagawa, H. Curr. Op. Chem. Biol. 2003, 13, 612-620;Bradbury, E. J.; Moon, L. D. F.; Popat, R. J.; King, V. R.; Bennett, G.S.; Patel, P. N.; Fawcett, J. W.; McMahon, S. B. Nature 2002, 416,636-640.). As with all glycosaminoglycans, the complexity andheterogeneity of Chondroitin sulfate (CS) glycosaminoglycans havehampered efforts to understand its precise biological roles. Forinstance, CS has been shown to prevent the growth of axons; yet it isalso found in developing, growth-permissive regions. (Bradbury et al.,supra; Emerling, D. E.; Lander, A. D. Neuron 1996, 17, 1089-1100). CSpolysaccharides have been shown both to stimulate and to attenuate thegrowth of cultured neurons. (Brittis, P. A. et al. Science 1992, 255,733-736; Dou, C. L.; Levine, J. M. J. Neurosci. 1995, 15, 8053-8066;Nadanaka, S. et al. J. Biol. Chem. 1998, 273, 3296-3307.) Notably, themolecules used in those studies were ˜200 saccharides in length, poorlydefined, and heterogeneously sulfated, features that might account forthe contradictory observations.

In addition, CS having a particular sulfation pattern (CS-E) is found onthe protein appican, an isoform of the amyloid precursor protein thatexhibits neurotrophic activity (Tsuchida, K. et al. J. Biol. Chem. 2001,276, 37155-37160). Moreover, polysaccharides enriched in the CS-E motifhave been shown to promote the outgrowth of neurons (Nadanaka, S. et al.J. Biol. Chem. 1998, 273, 3296-3307).

Because of the interest in studying neuronal growth and differentiation,there is a need for small molecule modulators of neuronal growth.

SUMMARY

Provided herein are small molecule compounds that are modulators ofneuronal growth. The compounds provided herein are oligosaccharides oroligosaccharide-like molecules having a plurality of negatively chargedmoieties. Also provided are compositions and methods of using thecompounds and compositions for the treatment of conditions associatedwith neurological disorders.

In certain embodiments, the compounds provided herein arepolysaccharides containing repeating dimer units of formula I:

wherein R¹, R², R³ and R⁴ are each independently selected from hydrogenor negatively charged groups, including but not limited to sulfate,phosphate and carboxylate; with a proviso that

a) at least one of R¹, R², R³ or R⁴ is not hydrogen;

b) when R¹ is sulfate, R² is not OH; and

c) when R² is sulfate, R¹ is not OH.

In certain embodiments, the compounds provided herein have formula II:

or pharmaceutically acceptable derivatives thereof,

wherein R¹, R², R³ and R⁴ are each independently selected from hydrogenor negatively charged groups, including but not limited to sulfate,phosphate and carboxylate; R⁶ is selected from optionally substitutedalkyl, optionally substituted alkenyl or optionally substituted alkynyl;and n is 0-100; provided that at least one of R¹, R², R³ and R⁴ is nothydrogen.

Pharmaceutically-acceptable derivatives, including salts, esters, enolethers, enol esters, solvates, hydrates and prodrugs of the compoundsdescribed herein are provided herein. Further provided arepharmaceutical compositions containing the compounds provided herein anda pharmaceutically acceptable carrier. In one embodiment, thepharmaceutical compositions are formulated for single dosageadministration.

Methods of treating, using the compounds and compositions herein areprovided. Methods of treating, preventing, or ameliorating one or moresymptoms associated with neurological disorders using the compounds andcompositions provided herein are provided. In practicing the methods,effective amounts of the compounds or compositions containingtherapeutically effective concentrations of the compounds areadministered. In certain embodiments, the compounds provided herein areused in conjunction with proteins or other factors to stimulate thegrowth of the implanted tissue. In certain embodiments, the compoundsprovided herein interact with growth factors and cytokines (e.g., tumornecrosis factor-α or TNFα and nerve growth factor or NGF).

Articles of manufacture are provided containing packaging material, acompound or composition provided herein which is useful for treating,preventing, or ameliorating one or more symptoms associated withneurological disorders using the compounds and compositions providedherein, and a label that indicates that the compound or composition isuseful for treating, preventing, or ameliorating one or more symptomsassociated with neurological disorders.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B illustrate the effect of disaccharide CS-E, tetrasaccharideCS-E, and unsulfated tetrasaccharide on neuronal morphology and growthof hippocampal neuron.

FIGS. 2A-B show the effect of disaccharide CS-E, tetrasaccharide CS-E,and unsulfated tetrasaccharide on the growth of dopaminergic neurons.

FIGS. 3A-B show the effect of disaccharide CS-E, tetrasaccharide CS-E,and unsulfated tetrasaccharide on the growth of dorsal root ganglion(DRG) neurons from the spinal cord.

FIG. 4 illustrates the effect of tetrasaccharides CS-E, CS-C, CS-R andCS-E dimer on neuronal growth of hippocampal neuron.

FIG. 5 illustrates binding selectivity of tetrasaccharides CS-E andCS-C, and dimer CS-E to tumor necrosis factor-α.

FIG. 6 shows the antagonizing effect of CS polysaccharides enriched inthe CS-E, CS-C and CS-A sulfation pattern on binding of TNFα to itsreceptor, as demonstrated by competition ELISA.

FIG. 7 illustrates binding of tetrasaccharides CS-E, CS-C anddisaccharide CS-E to neuronal growth factor midkine.

DETAILED DESCRIPTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications are incorporated byreference in their entirety. In the event that there are a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, the term “outgrowth” refers to the process by whichaxons grow out of a neuron. The outgrowth can result in a totally newaxon or the repair of a partially damaged axon.

As used herein, the term “CNS neurons” is intended to include theneurons of the brain and the spinal cord which are unresponsive to nervegrowth factor (NOF).

As used herein, the term “injury” is intended to include a damage whichdirectly or indirectly affects the normal functioning of the CNS. Forexample, the injury can be damage to retinal ganglion cells; a traumaticbrain injury; a stroke related injury; a cerebral aneurism relatedinjury; a spinal cord injury, including monoplegia, diplegia,paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorderor neuropathic pain syndrome.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs thereof. Such derivatives may be readily prepared by thoseof skill in this art using known methods for such derivatization. Thecompounds produced may be administered to animals or humans withoutsubstantial toxic effects and either are pharmaceutically active or areprodrugs. Pharmaceutically acceptable salts include, but are not limitedto, amine salts, such as but not limited toN,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia,diethanolamine and other hydroxyalkylamines, ethylenediamine,N-methylglucamine, procaine, N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, nitrates, borates, methanesulfonates, benzenesulfonates,toluenesulfonates, salts of mineral acids, such as but not limited tohydrochlorides, hydrobromides, hydroiodides and sulfates; and salts oforganic acids, such as but not limited to acetates, trifluoroacetates,maleates, oxalates, lactates, malates, tartrates, citrates, benzoates,salicylates, ascorbates, succinates, butyrates, valerates and fumarates.Pharmaceutically acceptable esters include, but are not limited to,alkyl, alkenyl, alkynyl, and cycloalkyl esters of acidic groups,including, but not limited to, carboxylic acids, phosphoric acids,phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.Pharmaceutically acceptable enol ethers include, but are not limited to,derivatives of formula C═C(OR) where R is hydrogen, alkyl, alkenyl,alkynyl, and cycloalkyl. Pharmaceutically acceptable enol estersinclude, but are not limited to, derivatives of formula C═C(OC(O)R)where R is hydrogen, alkyl, alkenyl, alkynyl, or cycloalkyl.Pharmaceutically acceptable solvates and hydrates are complexes of acompound with one or more solvent or water molecules, or 1 to about 100,or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

As used herein, glycopolymers refer to polymers with saccharide groupspendant to the main chain.

As used herein, “linker” refers to the intervening atoms between the twosaccharide groups or the saccharide group and the polymer chain. Thelinker is characterized by a first chemical functional group thatconnects the first end of the linker to a first saccharide and a secondchemical functional group that connects the second end of the linker toa second saccharide of to a polymer backbone.

As used herein, treatment means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use for treating conditionsassociated with neurological disorders.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular compound or pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe compound or composition provided herein.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized by one or more steps or processes orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, thepharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392). Other prodrugs for useherein are described elsewhere herein.

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is understood that the present inventionencompasses any racemic, optically active, polymorphic, or steroisomericform, or mixtures thereof, of a compound of the invention, whichpossesses the useful properties described herein, it being well known inthe art how to prepare optically active forms and how to determineantiproliferative activity using the standard tests described herein, orusing other similar tests which are well known in the art.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis, high performance liquid chromatography (HPLC) and massspectrometry (MS), used by those of skill in the art to assess suchpurity, or sufficiently pure such that further purification would notdetectably alter the physical and chemical properties, such as enzymaticand biological activities, of the substance. Methods for purification ofthe compounds to produce substantially chemically pure compounds areknown to those of skill in the art. A substantially chemically purecompound may, however, be a mixture of stereoisomers. In such instances,further purification might increase the specific activity of thecompound.

As used herein, the term “alkyl” refers to a monovalent straight orbranched chain or cyclic radical. In certain embodiments, the alkylgroup contains from one to twenty-four carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl,octadecyl, nonadecyl, eicosyl, 18-methyl-nonadecyl, 19-methyl-eicosyl,and the like. As used herein lower alkyl refers to alkyl groups of 1 to6 carbon atoms.

As used herein, “substituted alkyl” refers to alkyl groups furtherbearing one or more substituents, including, but not limited tosubstituents selected from lower alkyl, hydroxy, alkoxy (of a loweralkyl group), mercapto (of a lower alkyl group), cycloalkyl, substitutedcycloalkyl, heterocyclic, substituted heterocyclic, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy,halogen, trifluoromethyl, cyano, azido, nitro, nitrone, amino, amino,—C(O)H, acyl, oxyacyl, carboxyl, carbamate, sulfonyl, sulfonamide, andsulfuryl, which may be protected or unprotected as necessary, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Ed. 1991, hereby incorporated by reference.

As used herein, “alkenyl” refers to straight or branched chainhydrocarbon group having one or more carbon-carbon double bonds. Incertain embodiments, the alkenyl group contains from 2 up to 24 carbonatoms, and “substituted alkenyl” refers to alkenyl groups furtherbearing one or more substituents as set forth above.

As used herein, “alkynyl” refers to straight or branched chainhydrocarbon group having one or more carbon-carbon triple bonds. Incertain embodiments, the alkynyl group contains from 2 up to 24 carbonatoms, and “substituted alkynyl” refers to alkynyl groups furtherbearing one or more substituents as set forth above.

Where the number of any given substituent is not specified (e.g.,haloalkyl), there may be one or more substituents present. For example,“haloalkyl” may include one or more of the same or different halogens.

As used herein “subject” is an animal, typically a mammal, includinghuman, such as a patient.

The phrase “effective amount” as used herein means an amount requiredfor prevention, treatment, or amelioration of one or more of thesymptoms associated with neurological disorders. Effective amounts canbe measured by any methods known to one of skill in the art. In certainembodiments, effective amounts can be measured by improvements inneuronal or ganglion cell survival, axonal regrowth or neurodegenerationand connectivity following axotomy using well known methods. See, e.g.,Bray, et al., “Neuronal and Normeuronal Influences on Retinal GanglionCell Survival, Axonal Regrowth, and Connectivity After Axotomy”, Ann.N.Y. Acad. Sci., pp. 214-228 (1991).

As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intramuscular, intrathecal or intravitrealinjection, or infusion techniques.

The term “topically” encompasses administration rectally and byinhalation spray, as well as the more common routes of the skin andmucous membranes of the mouth and nose and in toothpaste.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

B. Compounds

In one embodiment, the compounds for use in the compositions and methodsprovided herein have formula:

or pharmaceutically acceptable derivatives thereof,

wherein R¹, R², R³ and R⁴ are each independently selected as follows:

i) R¹ and R² are each independently selected from sulfate, phosphate andcarboxylate; and R³ and R⁴ are hydrogen;

ii) R³ and R⁴ are each independently selected from sulfate, phosphateand carboxylate; and R¹ and R² are hydrogen;

iii) R¹, R² and R³ are each independently selected from sulfate,phosphate and carboxylate; and R⁴ is hydrogen;

iv) R¹, R² and R⁴ are each independently selected from sulfate,phosphate and carboxylate; and R³ is hydrogen;

v) R² and R⁴ are each independently selected from sulfate, phosphate andcarboxylate; and R¹ and R³ are hydrogen;

vi) R¹ is selected from sulfate, phosphate and carboxylate; and R², R³and R⁴ are hydrogen;

vii) R² is selected from sulfate, phosphate and carboxylate; and R¹, R³and R⁴ are hydrogen;

viii) R³ is selected from sulfate, phosphate and carboxylate; and R², R⁴and R¹ are hydrogen; or

ix) R⁴ is selected from sulfate, phosphate and carboxylate; and R¹, R²and R³ are hydrogen;

R⁵ is optionally substituted alkyl or optionally substituted alkenyl;and

n is 0-100.

In another embodiment, n is 0-50. In another embodiment, n is 0-25. Inanother embodiment, n is 0-20. In another embodiment, n is 0-15. Inanother embodiment, n is 0-10. In another embodiment, n is 0-5. Inanother embodiment, n is 0-4. In another embodiment, n is 2-4. Inanother embodiment, n is 4. In another embodiment, n is 3. In anotherembodiment, n is 2. In another embodiment, n is 1. In anotherembodiment, n is 0, 1, 2, 3 or 4.

In another embodiment, R⁵ is lower alkenyl. In another embodiment, R⁵ isallyl.

In another embodiment, R¹ is selected from hydrogen, sulfate, phosphateand carboxylate. In another embodiment, R¹ is sulfate.

In another embodiment, R² is selected from hydrogen, sulfate, phosphateand carboxylate. In another embodiment, R² is sulfate. In anotherembodiment, R¹ is hydrogen.

In another embodiment, R³ is selected from hydrogen, sulfate, phosphateand carboxylate. In another embodiment, R³ is sulfate. In anotherembodiment, R³ is hydrogen.

In another embodiment, R⁴ is selected from hydrogen, sulfate, phosphateand carboxylate. In another embodiment, R⁴ is sulfate. In anotherembodiment, R⁴ is hydrogen.

In one embodiment, the compounds provided herein have formula III:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula IV:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula V:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula VI:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula VI:

In one embodiment, the compounds provided herein have formula VII:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula VIII:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula IX:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula X:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein have formula XI:

wherein the variables are as described elsewhere herein.

In one embodiment, the compounds provided herein are selected from:

wherein the variables are as described elsewhere herein.

In one embodiment, the compound provided herein is selected from:

In one embodiment, the compound is selected from:

In one embodiment, the compound is

In certain embodiments, the compounds provided herein are substantiallypure. In certain embodiments, the negatively charged groups in thecompounds provided herein can be prepared as a protected derivative thatis unmasked in the biological milieu, e.g., an ester that can be cleavedto give a carboxyate.

In certain embodiments, the compounds provided herein arepolysaccharides containing repeating dimer units of formula I:

wherein R¹ and R² are sulfate and the other variables are as describedelsewhere herein. In certain embodiments, the compounds provided hereinare polysaccharides containing repeating dimer units of formula I,wherein R^(I) and R² are sulfate and R³ and R⁴ are hydrogen.

In certain embodiments, the compounds provided herein are linked withvarious linker moieties to be polyvalent. In one embodiment, the linkermoieties used are labile under biological conditions, such that uponexposure to biological conditions, the linker will be severed and willrelease the compound provided herein. Such linkers are known in the art.

In certain embodiments, the linkers are bifunctional molecules that canform a bond between the two saccharide moieties. The linker can behomobifunctional or heterobifunctional. Examples of functional groups onthe linker include, but are not limited to —NH₂, —ONH₂, —NHC═(O), —OH,—CHO, —CO₂H, and —SH. Each of these functional groups can form acovalent linkage between the two saccharide.

The linker can include linear or acyclic portions, cyclic portions,aromatic rings or combinations thereof. In certain embodiments, thelinker can have from 1 to 100 main chain atoms other than hydrogenatoms, selected from C, N, O, S, P and Si. In certain embodiments thelinker contains up to 50 main chain atoms other than hydrogen, up to 40,up to 30, up to 20, up to 15, up to 10, up to 5, up to 2 main chainatoms other than hydrogen. In certain embodiments the linker is acyclic.In certain embodiments, the linker contains oligomers of ethylene glycolor alkylene chains or mixtures thereof.

In certain embodiments, the linker has formula:

wherein n₁, is 1-100, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 2 or 1 and R is

Where the variables are as described elsewhere herein.

In certain embodiments, the compounds with linker contain the repeatingdisaccharide units linked via a linker. In certain embodiments, thecompound is dimer CS-E of formula:

In certain embodiments, the saccharide compounds with repeating units offormula I or compounds of formula II are copolymerized or grafted intopolymer backbonse to form glycopolymers. The polymer backbone in certainembodiments, is a straight chain, in other embodiments it is a branchedpolymer. In certain embodiments, the glycopolymer provided hereincontains between about 2 and about 1000 pendent saccharide moieties.

The saccharide compounds with repeating units of formula I or compoundsof formula II can be linked to the polymer backbone via linkers. Thelinkers are bifunctional molecules that can form a bond between thesaccharide and the polymer backbone. The linker can be homobifunctionalor heterobifunctional. Examples of functional groups on the linkerinclude, but are not limited to —NH₂, —ONH₂, —NHC═(O), —OH, —CHO, —CO₂H,and —SH. Each of these functional groups can form a covalent linkagebetween the saccharide and the polymer backbone.

The linker can include linear or acyclic portions, cyclic portions,aromatic rings or combinations thereof. In certain embodiments, thelinker can have from 1 to 100 main chain atoms other than hydrogenatoms, selected from C, N, O, S, P and Si. In certain embodiments thelinker contains up to 50 main chain atoms other than hydrogen, up to 40,up to 30, up to 20, up to 15, up to 10, up to 5, up to 2 main chainatoms other than hydrogen. In certain embodiments the linker is acyclic.In certain embodiments, the linker contains oligomers of ethylene glycolor alkylene chains or mixtures thereof.

The polymer backbones for use herein are known in the art. In certainembodiments, the polymer backbone is polyacrylamide based. In otherembodiments, the polymer is selected from polyacrylamide, polyacrylateand poly(N-acryloxy)succinimide.

In certain embodiments, the glycopolymers provided herein are selectedfrom:

wherein n₁, n₂, n₃ and n₄ are each independently 1-100, 1-50, 1-40,1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2 or 1 and theother variables are as described elsewhere herein.

C. Preparation of Compounds

The compounds provided herein can be prepared by using routine chemicalreactions known in the art. In certain embodiments, the compounds can beprepared by using a convergent strategy (illustrated in schemes 1-3) toaccess various CS-E molecules from a single disaccharide building block,4. The sulfated 4- and 6-hydroxyls of D-galactosamine were masked with ap-methoxybenzylidene acetal. This group allows access to other sulfationmotifs (e.g., CS-A, CS-C). In certain embodiments, the reactions can beextended to solid-phase methodologies. In particular, oxidative removalusing DDQ (Zhang, Z. Y.; Magnusson, G. J. Org. Chem. 1996, 61,2394-2400) or regioselective opening of the acetal ring (Tamura, J. etal. Carbohydr. Res. 1997, 305, 43-63; Johansson, R.; Samuelsson, B. J.Chem. Soc.-Perkin Trans. 1 1984, 2371-2374), in certain embodiments,permits selective deprotection of either or both the 4- and 6-hydroxylsand circumvents the need for hydrogenolysis. The orthogonaltert-butyldimethylsilyl group can be installed at the C-4 position onthe non-reducing end of 4 to facilitate chain elongation. To achievestereoselective formation of β-glycosidic linkages, N-trichloroacetyland benzoyl participating groups are used (Coutant, C.; Jacquinet, J. C.J. Chem. Soc.-Perkin Trans. 1 1995, 1573-1581). Finally, the anomerichydroxyl of 4 is masked with an allyl group, which could be converted toactivated glycosyl donors and offers a convenient means to conjugate CSto small molecules, proteins or surfaces.

The synthesis of the target compounds is illustrated in Scheme 1.Monosaccharides 5 and 6 were generated from knownp-tolyl-1-thio-13-D-glucopyranose (Clingman, A. L.; Richtmyer, N. K. J.Org. Chem. 1964, 29, 1782-1787.) or1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-D-galactopyranose (Lemieux, R.U.; Ratcliffe, R. M. Can. J. Chem.-Rev. Can. Chim. 1979, 57, 1244-1251),respectively (Schemes 2 and 3). Coupling of 5 with 6 using thetrichloroacetimidate procedure (Schmidt, R. R.; Kinzy, W. In Adv.Carbohydr. Chem. Biochem., 1994; Vol. 50, pp 21-123) affordedexclusively the β-linked disaccharide 4 in 74% yield. At this stage,activation of the disaccharide, in certain embodiments, proceeds throughconversion of the C-1 allyl group to the lactol. However, conventionalmethods such as PdCl₂, Pd(PPh₃)₄, [Ir(COD)(PMePh₂)₂]PF₆ and Wilkinson'scatalyst did not yield the desired isomerization, presumably due tointerference by the neighboring trichloroacetamide (a related2-acetamido derivative underwent isomerization readily upon treatmentwith [Ir(COD)(PMePh₂)₂]PF₆.) Treatment with Grubbs' second-generationcatalyst (Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett.1999, 1, 953-956) in the presence of H₂ led to the desired outcome.Hydrolysis of the enol ether and conversion to tichloroacetimidate 7proceeded smoothly under standard conditions (Driguez, P. A.; Lederman,I.; Strassel, J. M.; Herbert, J. M.; Petitou, M. J. Org. Chem. 1999, 64,9512-9520; Shiozaki, M et al. Carbohydr. Res. 1991, 222, 57-68).Disaccharide 4 also provided ready access to glycosyl acceptor 8 viadesilylation. Subsequent glycosylation of 7 and 8 delivered the desiredtetrasaccharide with good stereoselectivity.

The fully protected di- and tetrasaccharides were subjected to the finaldeprotection-sulfation steps. Radical-mediated conversion of theN-trichloroacetyl group into an N-acetyl (Coutant, C.; Jacquinet, J. C.J. Chem. Soc.-Perkin Trans. 1 1995, 1573-1581) followed by oxidativecleavage of the p-methoxybenzylidene acetal (Zhang, Z. Y.; Magnusson, G.J. Org. Chem. 1996, 61, 2394-2400.) afforded 9 and 10. Treatment of 9and 10 with SO₃.timethylamine complex in DMF delivered the sulfatedcompounds in 67% and 93% yield, respectively, see Tamura, J.; Neumann,K. W.; Kurono, S.; Ogawa, T. Carbohydr. Res. 1997, 305, 43-63. Thetarget CS-E di- and tetrasaccharides 1 and 2 were obtained after silyldeprotection and saponification utilizing NaOH or sequential LiOOH—NaOHtreatment (Lucas, H.; Basten, J. E. M.; Van Dinther, T. G.; Meuleman, D.G.; Van Aelst, S. F.; Van Boeckel, C. A. A. Tetrahedron 1990, 46,8207-8228) to minimize β-elimination at the C-4 position. Deprotectionof 9 under similar conditions furnished the unsulfated tetrasaccharide3.

^(α)a) TMSOTf, CH₂Cl₂, −40° C.→−15° C., 74%. (b) L(PCy₃)Cl₂Ru=CHPh,L=1,3-dimesitylimidazolylidene (20 mol %), H₂, 77%. (c) I₂, H₂O,pyr/THF, 81%. (d) DBU, CCl₃CN, CH₂Cl₂, 90%. (e) HF.pyr, pyr/THF, 0° C.,85%. (f) TMSOTf, CH₂Cl₂, −15° C., 31%. (g) TBTH, AIBN, DMA/benzene, 25°C.→80° C., 85%. (h) DDQ, 1120/CH₂O₂, 75%. (i) SO₃.Me₃N, DMF, 50° C.,67%. (j) HF.pyr, pyr/THF/H₂O, 0° C. (k) LiOH, H₂O₂, THF/H₂O, then NaOH,MeOH/H₂O, 25% over three steps. (1) TBTH, AIBN, benzene, 25° C.→80° C.,85%. (m) DDQ, H₂O/CH₂Cl₂, 62%. (n) SO₃Me₃N, DMF, 50° C., 93%. (o)HF.pyr, pyr/THF/H₂O, 0° C. (p) NaOH, MeOH/H₂O, 55% over two steps (q)HF.pyr, pyr/THF/H₂O, 0° C. (r) LiOH, H₂O₂, THF/H₂O, then NaOH, MeOH/H₂O,52% over three steps.

Scheme 4 represents synthesis of exemplary compounds provided herein:

The preparation of glycoconjugates provided herein can be represented asfollows:

a) Copolymerization of acrylamide with monomeric acrylamides in whichone of the acrylamide possesses the compound of formula I or IIdescribed herein.

Where the variables are as described elsewhere herein.

b1) Graft conjugation of nucleophilic ligands topoly[(N-acryloxy)succinimide] based on oxide linkers:

b2) Polyacryamide based polymers based on oxime linkers.

c1) Polyacrylamide based polymers based on 2-aminoethanethiol.

c2) Polyacrylamide based polymers based on 2-aminoethanethiol

d) Other polyacrylamide based polymers

e) Scheme 9 illustrates Grubbs metathesis approach towards glycopolymerswith an exemplary compound provided herein

Synthesis of saccharides linked via linkers provided herein isrepresented in schemes 10 and 11 as follows:

1. Oxime Linker:

where the variables are as described elsewhere herein.

2. Amine Linker

where the variables are as described elsewhere herein.

In certain embodiments, R is

D. Formulation of Pharmaceutical Compositions

The pharmaceutical compositions provided herein contain therapeuticallyeffective amounts of one or more of the compounds provided herein thatare useful in the prevention, treatment, or amelioration of one or moreof the symptoms associated with neurological disorders and apharmaceutically acceptable carrier. Pharmaceutical carriers suitablefor administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

The compositions contain one or more compounds provided herein. Thecompounds are, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, thecompounds described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof is (are) mixed with asuitable pharmaceutical carrier. The compounds may be derivatized as thecorresponding salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs prior to formulation, as described above. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates oneor more of the symptoms associated with neurological disorders. In oneembodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems well known tothose of skill in the art and then extrapolated therefrom for dosagesfor humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to ameliorate one or more of the symptomsassociated with neurological disorders, as described herein.

In one embodiment, a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.1 ng/ml toabout 50-100 μg/ml. The pharmaceutical compositions, in anotherembodiment, should provide a dosage of from about 0.001 mg to about 2000mg of compound per kilogram of body weight per day. Pharmaceuticaldosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment fromabout 10 mg to about 500 mg of the active ingredient or a combination ofessential ingredients per dosage unit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as TWEEN®, or dissolution in aqueous sodium bicarbonate.Derivatives of the compounds, such as prodrugs of the compounds may alsobe used in formulating effective pharmaceutical compositions.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

1. Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

a. Solid Compositions for Oral Administration

In certain embodiments, the formulations are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder; a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an emetic coating; and a film coating.Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, molasses,polyinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The compound, or pharmaceutically acceptable derivative thereof, couldbe provided in a composition that protects it from the acidicenvironment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. Thecomposition may also be formulated in combination with an antacid orother such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included. In all embodiments,tablets and capsules formulations may be coated as known by those ofskill in the art in order to modify or sustain dissolution of the activeingredient. Thus, for example, they may be coated with a conventionalenterically digestible coating, such as phenylsalicylate, waxes andcellulose acetate phthalate.

b. Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g., for example, in a polyethylene glycol, may be dilutedwith a sufficient quantity of a pharmaceutically acceptable liquidcarrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl)acetals of lower alkyl aldehydes such as acetaldehydediethyl acetal.

2. Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a compound providedherein is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous. Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone.

Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering orchelating agent of metal ions include EDTA. Pharmaceutical carriers alsoinclude ethyl alcohol, polyethylene glycol and propylene glycol forwater miscible vehicles; and sodium hydroxide, hydrochloric acid, citricacid or lactic acid for pH adjustment. The concentration of thepharmaceutically active compound is adjusted so that an injectionprovides an effective amount to produce the desired pharmacologicaleffect. The exact dose depends on the age, weight and condition of thepatient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active compound to thetreated tissue(s).

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

3. Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

4. Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

For nasal administration, the preparation may contain an esterifiedphosphonate compound dissolved or suspended in a liquid carrier, inparticular, an aqueous carrier, for aerosol application. The carrier maycontain solubilizing agents such as propylene glycol, surfactants,absorption enhancers such as lecithin or cyclodextrin, or preservatives.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

5. Compositions for other Routes of Administration

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and rectal administration,are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and5,860,957.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm. Tablets and capsules for rectaladministration are manufactured using the same pharmaceuticallyacceptable substance and by the same methods as for formulations fororal administration.

6. Targeted Formulations

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MDT's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

7. Articles of Manufacture

The compounds or pharmaceutically acceptable derivatives may be packagedas articles of manufacture containing packaging material, a compound orpharmaceutically acceptable derivative thereof provided herein, which iseffective for treatment, prevention or amelioration of one or moresymptoms associated with neurological disorders, within the packagingmaterial, and a label that indicates that the compound or composition,or pharmaceutically acceptable derivative thereof, is used for thetreatment, prevention or amelioration of one or more symptoms associatedwith neurological disorders.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment. A wide array of formulations ofthe compounds and compositions provided herein are contemplated as are avariety of treatments for any symptoms associated with neurologicaldisorders.

E. Evaluation of the Activity of the Compounds

The activity of the compounds provided herein can be assessed by methodsand assays known to one of skill in the art. For example, the biologicalactivity can be assessed in assays known for testing the activity ofchondroitin sulfate.

In certain embodiments, the activity of the compounds provided herein tomodulate neuronal growth, can be tested using cultured primaryhippocampal neurons. Primary hippocampal neurons are cultured onpoly-DL-ornithine-coated coverslips with or without each compound. After48 h, the neurons are fixed, immunostained with anti-tau antibodies, andexamined by confocal fluorescence microscopy. The effect of sulfatedtetrasaccharide CS-E on neuronal morphology and growth is illustrated inFIGS. 1A-B. The number of neurites emanating from the cell body wasenhanced, and the growth of the major extension was stimulated by39.3±3.6% relative to the poly-DL-ornithine control. In contrast,sulfated disaccharide and unsulfated tetrasaccharide had no significanteffect on neuronal outgrowth.

Effect of disaccharide CS-E, tetrasaccharides CS-E, and unsulfatedtetrasaccharide on the growth of dopaminergic neurons as well as dorsalroot ganglion (DRG) neurons from the spinal cord is illustrated in FIGS.2A-B and 4A-B, respectively. In certain embodiments, the CS-Etetrasaccharide promoted the outgrowth of cultured dopaminergic and DRGneurons by 30-40% relative to the untreated controls. FIGS. 5 and 7illustrate binding of tetrasaccharides CS-E, CS-C and disaccharide CS-Eto tumor necrosis factor-α and midkine, respectively.

F. Methods of Use

In one embodiment, the compounds provided herein are useful asmodulators of neuronal growth. Thus, provided herein are methods ofpromoting regeneration of an injured or severed nerve or nerve tissue,or promoting outgrowth in neuronal cells under a variety of neurologicalconditions requiring neuronal cell outgrowth. The methods includecontacting a neuronal cell, or an injured or severed nerve, with acompound provided herein in an amount effective to promote neuronaloutgrowth. The method may be carried out in vitro or in vivo.

In certain embodiments, the compounds are used in in vitro studies ofneuronal growth. Any of a variety of mammalian neuronal cells, includingneuronal cells from brain, CNS, peripheral nerves and the like, can betreated by the methods provided herein. In addition, the cells can befrom any of a variety of mammalian species, including human, mouse,chicken, and any other mammalian species, including the agriculturalstock and non-domesticated mammals. In certain embodiments, thecompounds are used to induce neuronal growth in cultured neurons,including, but not limited to cultured hippocampal neurons, dopaminergicneurons and dorsal root ganglion (DRG) neurons from the spinal cord. Incertain embodiments, the compounds provided herein are useful ininducing growth of differentiated neural stem cells prior toimplantation. In the case of Parkinson's disease, for instance,implanted tissue has promise as a replacement for dying dopaminergicneurons.

In certain embodiments, the methods provided herein are useful intreating peripheral nerve damage associated with physical or surgicaltrauma, infarction, bacterial or viral infection, toxin exposure,degenerative disease, malignant disease that affects peripheral orcentral neurons, or in surgical or transplantation methods in which newneuronal cells from brain, spinal cord or dorsal root ganglia areintroduced and require stimulation of neuronal outgrowth from theimplant and innervation into the recipient tissue. Such diseases furtherinclude but are not limited to CNS lesions, gliosis, Parkinson'sdisease, Alzheimer's disease, neuronal degeneration, and the like.

In other embodiment, the compounds are used in in vivo applications andas therapeutics for treating a variety of conditions, including, but notlimited to neurological disorders resulting from brain disorders orspinal cord trauma. In the case of spinal cord injury, the compoundsprovided herein, in certain embodiments, assist in the rebuilding ofdamaged axons.

In certain embodiments, the compounds provided herein modulate theactivity of fibroblast growth factors. In other embodiments, thecompounds provided herein are used in the treatment of stroke,Parkinson's, and other neurological diseases in conjunction withfibroblast growth factors.

In certain embodiments, the compounds provided herein are administeredin combination with proteins that either induce neuronal growth orinhibit neuronal growth. In certain embodiments, the compounds providedherein are used to stimulate the growth of an implanted tissue, inconjunction with proteins or other factors that stimulate the growth. Incertain embodiments, the compounds provided herein are administered incombination with tumor necrosis factor-a or TNFα and nerve growth factoror NGF. In certain embodiments, the compounds provided herein interactwith growth factors and cytokines (e.g., tumor necrosis factor-a or TNFαand nerve growth factor or NGF).

In certain embodiments, provided herein are methods of screening forsmall molecule inducers of neuronal growth. The methods involve applyingto a cultured neuron a small molecule bearing a plurality of negativelycharged groups, and determining the increase in axon length of a treatedversus an untreated cell. The compounds provided herein, in certainembodiments, cause an increase in mean axon length relative to anuntreated cell between about 1 to about 50%. In certain embodiments, theincrease in mean axon length caused by the compounds provided herein isgreater than about 1%, about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50%. In certainembodiments, the increase in mean axon length is greater than about 10%,in other embodiment, greater than 20% and in another embodiment, greaterthan 30% relative to an untreated cell.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES General Methods and Experimental Details

Unless stated otherwise, reactions were performed in flame-driedglassware under a nitrogen or an argon environment, using freshlydistilled solvents. All other commercially obtained reagents were usedas received. Thin-layer chromatography (TLC) was performed using E.Merck silica gel 60 F254 precoated plates (0.25 mm). Visualization ofthe developed chromatogram was performed by fluorescence quenching,cerium ammonium molybdate stain, or ninhydrin stain as necessary. ICNsilica gel (particle size 0.032-0.063 mm) was used for flashchromatography. Gel filtration chromatography (Sephadex® G-10 and G-25ultrafine) was used in order to achieve purification of the finalproducts.

¹H NMR and proton decoupling experiments were recorded on Varian Mercury300 (300 MHz) and Varian Mercury 600 (600 MHz) spectrometers and arereported in parts per million (5) relative to Me₄Si (0.0 ppm). Data for¹H are reported as follows: chemical shift (5 ppm), multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet), couplingconstant in Hz, and integration. ¹³C NMR spectra were obtained on aVarian Mercury 300 (75 MHz) spectrometer and are reported in terms ofchemical shift. IR spectra were recorded on a Perkin Elmer Paragon 1000spectrometer and are reported in terms of frequency of absorption(cm⁻¹). A JASCO P-1010 was used to measure optical rotation. Massspectra were obtained from the Protein/Peptide MicroAnalyticalLaboratory and the Mass Spectrometry Facility at the CaliforniaInstitute of Technology.

Example 1 A). Synthesis of the Glucuronic Acid Monomer 5

A1) p-Methylphenyl 4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside(2′). The procedure for the preparation of 2′ was adapted from Ye et al.(Ye, X.-S.; Wong, C.-H. J. Org. Chem. 2000, 65, 2410-2431)p-Methylphenyl-1-thio-β-D-glucopyranoside (Clingman, A. L.; Richtmyer,N. K. J. Org. Chem. 1964, 29, 1782-1787) 1′ (36.7 g, 128 mmol) wasdissolved in DMF (30.0 mL) and CH₃CN (300 mL). p-Anisaldehyde dimethylacetal (44.0 mL, 256 mmol) and DL-10-camphorsulfonic acid (6.00 g, 25.6mmol) were added. The reaction was stirred at rt for 12 h. The reactionwas quenched with TEA and concentrated to afford an orange syrup. Theproduct was purified by flash chromatography (50%→70% EtOAc:hexanes) toafford 2′ (36.3 g, 70%) as a white crystalline solid. R_(f) 0.26 (50%EtOAc:hexanes). [α]_(D) ²¹=−38 (c=1.0, CH₂Cl₂); IR (thin film on NaCl):ν=3447, 2869, 1614, 1518, 1250, 1104, 1084, 1033 cm⁻¹; ¹H NMR (300 MHz,CDCl₃): δ=7.43 (d, J=8.1 Hz, 2H, SC₆H₄Me), 7.39 (d, J=9.0 Hz, 2H,C₆H₄OMe), 7.15 (d, J=7.5 Hz, 2H, SC₆H₄Me), 6.88 (d, J=9.0 Hz, 2H,C₆H₄OMe), 5.48 (s, 1H, MeOPhCH), 4.56 (d, J=9.9 Hz, 1H, H-1), 4.35 (dd,J=3.9, 10.5 Hz, 1H), 3.85-3.72 (m, 5H), 3.50-3.39 (m, 3H), 2.80 (br s,1H, OH), 2.67 (br s, 1H, OH), 2.36 (s, 3H, SPhCH₃); ¹³C NMR (75 MHz,CDCl₃): δ=138.8, 138.2, 133.6, 132.1, 129.9, 129.4, 127.7, 113.7, 101.8,88.7, 80.2, 74.5, 72.5, 70.5, 68.6, 55.3, 21.2; FAB MS: m/z: calcd forC₂₁H₂₅O₆S: 405.1372; found: 405.1359 [M+H]⁺.

A2) p-Methylphenyl2,3-di-O-benzoyl-4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside(3′). 2′ (23.7 g, 58.6 mmol) was dissolved in CH₂Cl₂ (670 mL). In aseparate flask, benzoyl chloride (17.0 mL, 146 mmol) was added dropwiseto a solution of 4-(dimethylamino)pyridine (DMAP, 25.1 g, 205 mmol) inCH₂Cl₂ (225 mL). The benzoyl chloride/DMAP solution was then slowlyadded to the solution of 2′. An additional volume of CH₂Cl₂ (19.0 mL)was used to complete the transfer of solution. The reaction was allowedto stir at rt for 25 min and then quenched with saturated aqueousNaHCO₃. The aqueous layer was extracted with CH₂Cl₂ (2×). The combinedorganic layers were washed with brine, dried over Na₂SO₄, filtered, andconcentrated to yield a pale yellow solid. This crude material waswashed with MeOH and crystallization from EtOAc afforded 3′ as a whitesolid (30.8 g, 86%). R_(f) 0.43 (30% EtOAc:hexanes). [α]_(D) ²²=+25(c=0.42, CH₂Cl₂); IR (thin film on NaCl): ν=2934, 1740, 1735, 1730,1715, 1700, 1617, 1614, 1517, 1272, 1251, 1095 cm⁻¹; ¹H NMR (300 MHz,CDCl₃): δ=7.98-7.90 (m, 4H, ArH), 7.56-7.30 (m, 10H, ArH), 7.12 (d,J=8.1 Hz, 2H, SC₆H₄Me), 6.82 (d, J—, 8.7 Hz, 2H, C₆H₄OMe), 5.76 (dd,J=9.3, 9.3 Hz, 1H, H-3), 5.49 (s, 1H, MeOPhCH), 5.43 (dd, J=9.3, 9.3 Hz,1H, H-2), 4.95 (d, J=10.5 Hz, 1H, H-1), 4.43 (dd, J=4.5, 10.8 Hz, 1H),3.90-3.82 (m, 2H), 3.76-3.67 (m, 4H), 2.35 (s, 3H, SPhCH₃); ¹³C NMR (75MHz, CDCl₃): δ=165.6, 165.2, 160.1, 138.8, 133.8, 133.3, 133.1, 129.9,129.8, 129.8, 129.4, 129.3, 129.2, 128.4, 128.3, 127.9, 127.5, 113.6,101.5, 87.3, 78.5, 73.4, 71.1, 71.0, 68.5, 55.3, 21.3; FAB MS: m/z:calcd for C₃₅H₃₃O₈S: 613.1896; found: 613.1879 [M+H]⁺.

A3) p-Methylphenyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside(4′). The procedure for the regioselective ring opening of 3′ wasadapted from Johansson et al. (Johansson, R.; Samuelsson, B. J. Chem.Soc, Perkin Trans. 1 1984, 2371-2374) 3′ (12.0 g, 19.6 mmol) wascombined with sodium cyanoborohydride (6.15 g, 97.9 mmol), activated 3 Åpowdered molecular sieves (12.0 g), and dissolved in DMF (261 mL). Thereaction was cooled to 0° C. Trifluoroacetic acid (15.3 mL, 196 mmol)was added dropwise to the reaction. The reaction was stirred at 0° C.for 1 h, and then allowed to warm to rt. The reaction stirred at rt for1 d. It was then filtered, diluted with CH₂Cl₂, and quenched with coldsaturated aqueous NaHCO₃. The aqueous layer was separated and extractedwith CH₂Cl₂ (2×). The combined organic layers were washed with saturatedaqueous NaHCO₃ (1×) and brine (1×), dried over Na₂SO₄, filtered, andconcentrated. To remove the remaining sodium cyanoborohydride, the crudematerial was re-dissolved in CH₂Cl₂ (250 mL) and washed with brine (3×).The organic layer was dried over Na₂SO₄, filtered, and concentrated toafford a white solid containing the desired alcohol. R_(f) 0.23 (30%EtOAc:hexanes).

The crude alcohol was dissolved in CH₂Cl₂ (476 mL), TEA (8.20 mL, 58.6mmol) was added, and the reaction cooled to 0° C.tert-Butyldimethylsilyl trifluoromethanesulfonate (11.2 mL, 48.8 mmol)was added dropwise to the reaction. The reaction was allowed to warm tort and stirred for 3 h. It was then quenched with saturated aqueousNaHCO₃ and diluted with CH₂Cl₂. The aqueous layer was separated andextracted with CH₂Cl₂ (3×). The combined organic layers were washed withbrine, dried over Na₂SO₄, filtered, and concentrated to afford an orangesyrup. The product was purified by flash chromatography (10%→12%EtOAc:hexanes) to afford 4′ (13.2 g, 94%) as a white foam. R_(f) 0.64(30% EtEtOAc:hexanes). [α]_(D) ²²=+36 (c=1.0, CH₂Cl₂); IR (thin film onNaCl): ν=2953, 2928, 2856, 1734, 1612, 1602, 1513, 1451, 1272, 1251,1106, 1089, 1069 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.92-7.87 (m, 4H,ArH), 7.51-7.27 (m, 10H, ArH), 7.03 (d, J=7.8 Hz, 21-1, SC₆H₄Me),6.94-6.91 (m, 2H, ArH), 5.59 (dd, J=9.2, 9.2 Hz, 1H, H-3), 5.30 (dd,J=9.6, 9.6 Hz, 1H, H-2), 4.88 (d, J=9.6 Hz, 1H, H-1), 4.60 (d, J=11.4Hz, 1H, CH₂PhOMe), 4.51 (d, J=11.7 Hz, 1H, CH₂PhOMe), 4.01 (dd, J=9.0,9.0 Hz, 1H, H-4), 3.84-3.64 (m, 6H, H-5, 11-6, H-6, PhOCH₃), 2.32 (s,3H, SPhCH₃), 0.74 (s, 9H, (CH₃)₃CSi), 0.02 (s, 3H, CH₃Si), −0.22 (s, 3H,CH₃Si); ¹³C NMR (75 MHz, CDCl₃): δ=165.9, 165.3, 159.2, 138.2, 133.4,133.1, 133.0, 130.5, 129.9, 129.9, 129.8, 129.7, 129.5, 129.3, 128.6,128.4, 128.3, 113.9, 86.1, 81.0, 77.5, 73.3, 71.3, 69.4, 68.7, 55.5,25.9, 21.5, 18.1, −3.9, −4.4; FAB MS: m/z: calcd for C₄₁H₄₇O₈SSi:727.2785; found: 727.2761 [M]⁺.

A4) p-Methylphenyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-1-thio-β-D-glueopyranoside(5′). In a flask covered with aluminum foil, 4′ (13.2 g, 18.1 mmol) wasdissolved in CH₂Cl₂ (440 mL). Water (23.0 mL) and2,3-dichloro-5,6-dicyano-1,4-benzoquinone (4.93 g, 21.7 mmol) wereadded. The reaction was stirred at rt for 13 h. The reaction was thenquenched with aqueous NaHCO₃, and water was added to dissolve allsolids. The aqueous layer was separated and extracted with CH₂Cl₂ (3×).The combined organic layers were washed with brine, dried over Na₂SO₄,filtered, and concentrated to yield a peach solid. The product waspurified by flash chromatography (40% CH₂Cl₂:hexanes→100% CH₂Cl₂→10%EtOAc: CH₂Cl₂) to afford 5′ (9.42 g, 86%) as a white foam. R_(f) 0.41(20% EtOAc:hexanes). [α]_(D) ²²=+62 (c=1.0, CH₂Cl₂); IR (thin film onNaCl): ν=3442, 2951, 2928, 2856, 1733, 1602, 1493, 1451, 1273, 1088,1070, 1027 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.92-7.88 (m, 4H, ArH),7.52-7.45 (m, 2H, ArH), 7.38-7.32 (m, 6H, ArH), 7.12 (d, J=8.1 Hz, 2H,SC₆H₄Me), 5.62 (dd, J=9.3, 9.3 Hz, 1H, H-3), 5.29 (dd, J=9.6, 9.6 Hz,1H, H-2), 4.93 (d, J=9.9 Hz, 1H, H-1), 4.02-3.92 (m, 2H), 3.81-3.73 (m,1H), 3.60-3.55 (d, J=11.4 Hz, 1H), 2.35 (s, 3H, SPhCH₃), 1.95 (br s, 1H,OH), 0.76 (s, 9H, (CH₃)₃CSi), 0.07 (s, 3H, CH₃Si), −0.20 (s, 3H, CH₃Si);¹³C NMR (75 MHz, CDCl₃): δ=165.9, 165.4, 138.7, 133.5, 133.3, 133.2,130.0, 130.0, 129.9, 129.8, 129.4, 128.5, 128.5, 128.4, 86.4, 81.1,77.2, 71.3, 69.0, 62.0, 25.9, 21.6, 18.2, −3.9, −4.3; FAB MS: m/z: calcdfor C₃₃H₄₁O₇SSi: 609.2342; found: 609.2321 [M+H]⁺.

A5) p-Methylphenyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-1-thio-β-D-glueopyranosyluronate(6′). 5′ (9.42 g, 15.5 mmol) was dissolved in DMF (115 mL). Pyridiniumdichromate (34.9 g, 92.8 mmol) was added, and the reaction was stirredat rt for 3 d. To precipitate and remove the chromium salts, EtOAc wasadded, and the reaction was filtered and concentrated (3×). Theremaining salts were removed by flash chromatography (100% EtOAc) toyield a white foam containing the desired carboxylic acid. R_(f) 0.17(30% EtOAc:hexanes).

The crude acid was dissolved in CH₂Cl₂ (187 mL) and cooled to 0° C.Diazomethane (93.0 mL, 0.2 M in diethyl ether, 18.6 mmol) was slowlyadded. The reaction stirred at 0° C. for 1 h. Glacial acetic acid wasadded to quench the reaction. It was then concentrated and purified byflash chromatography (10%→15% EtOAc:hexanes) to yield 6′ (6.04 g, 61%)as a white solid. R_(f) 0.67 (30% EtOAc:hexanes). [α]_(D) ²²=+54 (c=1.0,CH₂Cl₂); IR (thin film on NaCl): ν=3443, 2953, 2928, 2857, 1732, 1601,1493, 1451, 1437, 1269, 1085, 1069 cm⁻¹; ¹H NMR (300 MHz, CDCl₃):δ=7.90-7.86 (m, 4H, ArH), 7.52-7.46 (m, 2H, ArH), 7.38-7.31 (m, 6H,ArH), 7.10 (d, J=8.1 Hz, 2H, SC₆H₄Me), 5.59 (dd, J=9.3, 9.3 Hz, 1H,H-3), 5.30 (dd, J=9.6, 9.6 Hz, 1H, H-2), 4.90 (d, J=9.9 Hz, 1H, H-1),4.26 (dd, J=9.2, 9.2 Hz, 1H, H-4), 4.08 (d, J=8.7 Hz, 1H, H-5), 3.82 (s,31-1, CO₂CH₃), 2.33 (s, 3H, SPhCH₃), 0.71 (s, 9H, (CH₃)₃CSi), −0.05 (s,3H, CH₃Si), −0.22 (s, 3H, CH₃Si); ¹³C NMR (75 MHz, CDCl₃): δ=168.3,168.3, 165.9, 165.3, 138.8, 133.7, 133.4, 133.4, 130.0, 130.0, 130.0,129.7, 129.5, 128.5, 128.2, 87.2, 80.4, 76.6, 70.9, 70.7, 52.8, 25.6,21.4, 18.0, −4.2, −4.9; FAB MS: m/z: calcd for C₃₄H₄₁O₈SSi: 637.2291;found: 637.2284 [M+H]

A6) Methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-α/β-D-glucopyranosyluronate(7′). 6′ (6.09 g, 9.56 mmol) was dissolved in CH₂Cl₂ (67.0 mL) and water(0.700 mL) was added. A solution was prepared containing 2.93 gN-iodosuccinimide, 127 mL CH₂Cl₂, 3.10 mL THF, and 78.0 μL triflic acid.130 mL of this solution was added to the reaction mixture. The reactionstirred at rt. for 5.5 h. It was then quenched with 1 M Na₂S₂O₃ anddiluted with CH₂Cl₂. The aqueous layer was separated and extracted withCH₂Cl₂ (3×). The combined organic layers were washed with brine, driedover Na₂SO₄, filtered, and concentrated. The product was purified byflash chromatography (15%→30% EtOAc:hexanes) to afford 7′ (4.27 g, 84%,6.2β:1α) as a white foam. R_(f) 0.30, 0.36 (30% EtOAc:hexanes). [α]_(D)²²=+99 (c=1.0, CH₂Cl₂); IR (thin film on NaCl): ν=3455, 2954, 2930,2857, 1732, 1602, 1451, 1275, 1110, 1070 cm⁻¹; ¹H NMR (300 MHz, CDCl₃):δ=8.18-8.07 (m, 4H, ArH), 7.99-7.90 (m, 4H, ArH), 7.69-7.31 (m, 12H,ArH), 6.55 (d, J=3.3 Hz, 1H, H-1, α), 5.94 (dd, J=9.0, 9.9 Hz, 1H),5.72-5.58 (m, 3H), 5.22-5.14 (m, 2H), 4.62 (d, J=9.3 Hz, 1H, H-1, 13),4.40-4.27 (m, 2H), 4.13 (d, J=9.3 Hz, 1H), 3.81 (s, 3H, CO₂CH₃), 3.80(s, 3H, CO₂CH₃), 3.46 (d, J=3.6 Hz, 1H), 0.76 (s, 9H, (CH₃)₃CSi), 0.75(s, 9H, (CH₃)₃CSi), −0.01 (s, 6H, CH₃Si), −0.15 (s, 6H, CH₃Si); ¹³C NMR(75 MHz, CDCl₃): δ=169.8, 169.0, 168.7, 167.4, 167.3, 166.1, 165.9,165.0, 134.2, 133.9, 133.8, 133.6, 133.4, 130.3, 130.2, 130.1, 129.9,129.1, 129.0, 128.8, 128.6, 128.6, 92.2, 90.9, 75.8, 74.8, 74.6, 74.6,72.5, 72.4, 72.3, 71.1, 70.5, 70.2, 52.9, 25.7, 25.6, 18.0, −4.2, −4.9;FAB MS: m/z: calcd for C₂₇H₃₅O₉Si: 531.2050; found: 531.2041 [M+H]⁺.

A7) Methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-α-D-glocopyranosyluronatetrichloroacetimidate (5). The preparation of 5 was performed by using aprocedure modified from Driguez et. al. (Driguez, P.-A.; Lederman, I.;Strassel, J.-M.; Herbert, J.-M.; Petitou, M. J. Org. Chem. 1999, 64,9512-9520) 7′ (3.32 g, 6.26 mmol) was coevaporated with toluene (2×20mL) and dried under vacuum overnight. It was then dissolved in CH₂Cl₂(49.0 mL). Trichloroacetonitrile (3.80 mL, 37.5 mmol) and Cs₂CO₃ (0.820g, 2.50 mmol) were added. After stirring at rt for 4 h, additionaltrichloroacetonitrile (0.950 mL, 9.50 mmol) and Cs₂CO₃ (0.200 g, 0.600mmol) were added. The reaction was allowed to stir an additional 4 h andthen concentrated. The product was purified by flash chromatography (10%EtOAc:hexanes+0.1% TEA) to afford 5 (3.77 g, 89%), with a trace amountof the 13 anomer, as a white foam. R_(f) 0.57 (30% EtOAc:hexanes).[α]_(D) ²²=+99 (c=1.0, CH₂Cl₂); IR (thin film on NaCl): ν=3343, 2954,2930, 2858, 1757, 1735, 1676, 1602, 1451, 1315, 1267, 1111, 1095 cm⁻¹;¹H NMR (300 MHz, CDCl₃): δ=8.60 (s, 1H, C═NH), 7.96-7.87 (m, 4H, ArH),7.53-7.29 (m, 6H, ArH), 6.74 (d, J=3.9 Hz, 1H, H-1), 5.99 (dd, J=9.0,10.2 Hz, 1H, H-3), 5.43 (dd, J=3.9, 10.5 Hz, 1H, H-2), 4.51 (d, J=9.3Hz, 1H, H-5), 4.38 (dd, J=9.3, 9.3 Hz, 1H, H-4), 3.81 (s, 3H, CO₂CH₃),0.74 (s, 9H, (CH₃)₃CSi), −0.01 (s, 3H, CH₃Si), −0.15 (s, 3H, CH₃Si); ¹³CNMR (75 MHz, CDCl₃): δ=168.7, 165.7, 165.7, 160.8, 133.7, 133.5, 130.1,129.9, 129.7, 128.7, 128.6, 128.6, 93.4, 74.6, 72.5, 70.9, 70.8, 53.0,53.0, 25.7, 18.0, −4.1, −4.9; ESI MS: m/z: calcd for C₂₉H₃₄Cl₃NNaO₉Si:696.1; found: 696.2 [M+Na]⁺.

B) Synthesis of the Galactosamine Monomer 6

B1)1,3,4,6-tetra-O-acetyl-2-deoxy-2-trichloroacetamido-α/β-D-galactopyranoside(9′). 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-D-galactopyranoside(Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem.i Rev. Can. Chim. 1979,57, 1244-1251) 8′ (0.100 g, 0.268 mmol) in THF (5.00 mL), was addedp-tosic acid monohydrate (0.051 g, 0.27 mmol) followed by Pd/C (0.017 g,6 mol %). The reaction was then placed under an atmosphere of H₂ andstirred at rt for 18 h. The Pd/C was removed by filtration throughCelite and the solvent concentrated to afford an anomeric mixture ofcrude amines as a pale yellow foam. The crude mixture was used for thenext step without purification. To a solution of crude amines in THF (5mL), cooled to 0° C. was added trichloroacetylchloride (0.220 g, 1.21mmol, 0.130 mL) followed by TEA (0.180 g, 1.79 mmol, 0.250 mL). Thereaction mixture was stirred at 0° C. for 15 min and then quenched withsaturated aqueous NaHCO₃. The water layer was separated and extractedwith CH₂Cl₂ (2×) and the combined organics dried over Na₂SO₄ and thesolvent removed in vacuo to afford a yellow oil. Purification of thisoil by flash chromatography (30%→40% EtOAc:hexanes) afforded 9′ (0.099g, 75%, 3.1β:1αa) as a white solid R_(f) 0.61 and 0.53 (60%EtOAc:hexanes). ¹H NMR (300 MHz, CDCl₃): δ=6.73 (d, J=9.0 Hz, 2H, NH),6.30 (d, J=3.9 Hz, 1H, H-1, a), 5.45 (d, J=3.3 Hz, 3H), 5.32 (dd, J=3.5Hz and 11.3 Hz, 2H), 4.58 (m, 2H), 4.26 (dd, J=6.6 Hz, 6.6 Hz, 2H),4.20-4.03 (m, 4H), 2.17 (s, 6H), 2.15 (s, 6H), 2.02 (s, 6H), 2.00 (s,6H); ¹³C NMR (75 MHz, CDCl₃): δ=171.2, 170.5, 170.2, 168.7, 162.2, 90.5,69.0, 67.8, 66.8, 61.5, 49.6, 21.2, 21.0; ESI MS: m/z: calcd forC₁₆H₁₉C₁₃NO₁₀: 490.0075; found: 490 [M−H]⁻.

B2) p-Methylphenyl2-deoxy-2-trichloroacetamido-3,4,6-tri-O-acetyl-1-thio-β-D-galactopyranoside(10′). To a solution of 9′ (0.050 g, 0.10 mmol) in dry CH₂Cl₂ (0.35 mL)was added p-toluenethiol (0.042 g, 0.34 mmol) followed by BF₃.OEt₂(0.043 g, 0.30 mmol, 38 μL) and the reaction mixture stirred at rt.After 2 h, a further addition of p-toluenethiol (0.012 g, 0.10 mmol) andBF₃.OEt₂ (0.014 g, 0.10 mmol, 13 mL) was made followed by stirring at rtfor 1 h. The reaction mixture was quenched with saturated aqueous NaHCO₃and the organic phase washed twice with saturated aqueous NaHCO₃ andwater. The aqueous layers were back extracted with CH₂Cl₂ (3×) and thecombined organics washed with brine and dried over Na₂SO₄ to afford anamber oil. Purification of this oil by flash chromatography (20%→25%EtOAc:hexanes) afforded 10′ (0.044 g, 80%) as a white solid. R_(f) 0.51(50% EtOAc:hexanes). [α]_(D) ²³=−2.4 (c=0.5, CH₂Cl₂); IR (thin film onNaCl): ν=3450, 1752, 1655, 1529, 1493, 1370, 1230, 1082, 1045 cm⁻¹; ¹HNMR (300 MHz, CDCl₃): δ=7.42 (d, J=8.3 Hz, 2H, SC₆H₄Me), 7.12 (d, J=8.3Hz, 2H, SC₆H₄Me), 6.77 (d, J=8.7 Hz, 1H, NH), 5.39 (d, J=3.3 Hz, 1H,H-4), 5.29 (dd, J=3.3, 11.1 Hz, 1H, H-3), 4.89 (d, J=10.5 Hz, 1H, H-1),4.22-4.09 (m, 3H, H-2, H-6), 3.94 (dd, J=6.6, 6.6 Hz, 1H, H-5), 2.34 (s,3H, SPhCH₃), 2.13 (s, 3H, OC(O)CH₃), 2.04 (s, 3H, OC(O)CH₃), 1.97 (s,3H, OC(O)CH₃); ¹³C NMR (75 MHz, CDCl₃): δ=170.6, 170.5, 170.2, 161.9,138.8, 133.5, 129.9, 128.5, 92.5, 87.2, 74.9, 70.9, 67.1, 62.0, 51.7,21.6, 21.1, 21.0, 20.9; FAB MS: m/z: calcd for C₂₁H₂₅C₁₃NO₈S: 556.0367;found: 556.0369 [M+H]⁺.

B3)_(p)-Methylphenyl2-deoxy-2-trichloroacetamido-3-O-triisopropylsilyl-4,6-O-p-methoxybenzylidene-1-thio-β-D-galactopyranoside(11′). A solution of 10′ (17.9 g, 0.0320 mol) in dry CH₂Cl₂ (85 mL) andMeOH (435 mL) was stirred at rt for 30 min and NaOMe (25 wt % solutionin MeOH, 0.517 g, 9.58 mmol, 2.07 mL) was then added. The mixture wasstirred for 2 h and DOWEX 50×8-200 added and stirring continued for afurther 30 min. The DOWEX was removed by filtration and the solventremoved in vacuo to afford 11′ (13.5 g, 98%) as a yellow solid. Thiscompound was suitable for the next step without purification.

B4) p-Methylphenyl2-deoxy-2-triehloroacetamido-4,6-O-p-methoxybenzylidene-1-thio-β-D-galactopyranoside(12′). To a solution of 11′ (13.5 g, 0.0310 mol) in acetonitrile (800mL, minimum amount) was added p-anisaldehyde dimethyl acetal (11 g,0.063 mol, 12 mL) and DL-10-camphorsulfonic acid (10 mol %) and themixture stirred at rt for 12 h. The reaction mixture was quenched withTEA and the solvent concentrated to afford a yellow solid. Purificationof this solid by flash chromatography (40%→80% EtOAc:hexanes) afforded12′ (13 g, 76%) as a white solid. R_(f) 0.25 (50% EtOAc:hexanes).[α]_(D) ²⁴=−14.6 (c=0.5, CH₂Cl₂); IR (thin film on NaCl): ν=3333, 1687,1615, 1519, 1492, 1403, 1364, 1301, 1248, 1167, 1095, 1055 cm⁻¹; ¹H NMR(300 MHz, CDCl₃): δ=7.55 (d, J=8.4 Hz, 2H, SC₆H₄Me), 7.34 (d, J=8.7 Hz,2H, C₆H₄OMe), 7.12 (d, J=8.4 Hz, 2H, SC₆H₄Me), 6.88 (d, J=8.7 Hz, 2H,C₆H₄OMe), 6.81 (d, J=7.5 Hz, 1H, NH), 5.48 (s, 1H, MeOPhCH), 5.03 (d,J=9.9 Hz, 1H, H-1), 4.37 (dd, J=1.5, 12.6 Hz, 1H, H-6), 4.20-4.10 (m,2H, H-3, H-4), 4.01 (dd, J=1.5, 12.6 Hz, 1H, H-6), 3.83 (s, 3H, PhOCH₃),3.69 (m, 1H, H-2), 3.57 (s, 1H, H-5), 2.58 (d, J=10.5 Hz, 1H, OH), 2.37(s, 31-1, SPhCH₃); ¹³C NMR, (75 MHz, CDCl₃): δ=162.1, 160.5, 139.0,134.7, 130.2, 130.0, 128.1, 126.9, 113.8, 101.4, 84.0, 75.2, 70.7, 70.3,69.5, 55.7, 54.4, 21.7; FAB MS: m/z: calcd for C₂₃H₂₅Cl₃NO₆S: 548.0469;found: 548.0448 [M+H]⁺.

B5) p-Methylphenyl2-deoxy-2-trichloroacetamido-3-O-triisopropylsilyl-4,6-O-p-methoxybenzylidene-β-D-galactopyranoside(13′). To a solution of 12′ (5.6 g, 0.010 mol) in dry DMF (50 mL) at rtwas added triisopropylsilyl chloride (6.3 g, 0.033 mol, 7.0 mL),imidazole (2.7 g, 0.040 mol) and DMAP (0.49 g, 40 mol %). The reactionmixture was stirred for 4 h whereupon further addition oftriisopropylsilyl chloride (3.2 g, 0.016 mol, 3.5 mL), imidazole (1.4 g,0.020 mol) and DMAP (0.25 g, 20 mol %) were added. The reaction mixturewas stirred for 12 h and quenched with saturated aqueous NaHCO₃. Theaqueous layer was extracted with EtOAc (3×) and the combined organicswashed with brine and dried over MgSO₄ to afford a pale yellow oil.Purification of this oil by flash chromatography (10%→15% EtOAc:hexanes)afforded 13′ (5.3 g, 75%) as a white solid. R_(f) 0.57 (30%EtOAc:hexanes). [α]_(D) ²³=+5.9 (c=0.5, CH₂Cl₂); ER (thin film on NaCl):ν=2943, 2866, 1705, 1616, 1519, 1493, 1464, 1365, 1249, 1170, 1139,1083, 1051 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.57 (d, J=8.1 Hz, 2H,SC₆H₄Me), 7.38 (d, J=8.7 Hz, 2H, C₆H₄OMe), 7.07 (d, J=8.1 Hz, 2H,SC₆H₄Me), 6.87 (d, J=8.7 Hz, 2H, C₆H₄OMe), 6.85 (m, 1H, NH), 5.45 (s,1H, MeOPhCH), 5.39 (d, J=9.9 Hz, 1H, H-1), 4.62 (dd, J=3.2, 10.2 Hz, 1H,H-3), 4.37 (dd, J=1.7, 12.5 Hz, 1H, H-6), 4.13 (d, J=3.2 Hz, 1H, H-4),4.01 (dd, J=1.7, 12.5 Hz, 1H, H-1-6), 3.83 (s, 3H, PhOCH₃), 3.71 (m, 1H,H-2), 3.55 (s, 1H, H-5), 2.34 (s, 3H, SPhCH₃), 1.01 (s, 21H,[(CH₃)₂CH]₃), ¹³C NMR (75 MHz, CDCl₃): δ=161.3, 160.1, 138.5, 134.1,130.7, 130.0, 128.0, 127.9, 113.5, 101.1, 83.4, 76.7, 71.0, 70.3, 69.7,55.6, 54.8, 21.7, 18.5, 18.4, 13.1; FAB MS: m/z: calcd forC₃₂H₄₅Cl₃NO₆SSi: 704.1621; found: 704.1623 [M+H]⁺.

B6) Allyl2-deoxy-2-trichloroacetamido-3-O-triisopropylsilyl-4,6-O-p-methoxybenzylidene-β-D-galactopyranoside(14′). To a solution of 13′ (11 g, 0.016 mol) in dry CH₂Cl₂ (675 mL) wasadded 4 Å powdered molecular sieves and the mixture stirred for 1 h.Allyl alcohol (9.3 g, 0.16 mol, 11 mL) and N-iodosuccinimide (5.3 g,0.023 mol) was added and the mixture cooled to 0° C. Triflic acid (0.5 Nsolution in CH₂Cl₂, 1.44 g, 9.60 mmol, 19.2 mL) was added and thereaction stirred at 0° C. for 10 min. The mixture was quenched with TEA,washed with brine and dried over MgSO₄. The solvent was removed in vacuoto afford a yellow oil. Purification of this oil by flash chromatography(5%→15% EtOAc:hexanes) afforded 14′ (8.1 g, 79%) as a white solid. R_(f)0.41 (30% EtOAc:hexanes). [α]_(D) ²⁴=+38.1 (c=0.5, CH₂Cl₂); IR (thinfilm on NaCl): ν=3445, 1644, 1520, 1463, 1368, 1249, 1171, 1123, 1060cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.45 (d, J=8.9 Hz, 2H, C₆H₄OMe), 6.97(d, J=7.2 Hz, 1H, NH), 6.87 (d, J=8.9 Hz, 2H, C₆H₄OMe), 5.96-5.83 (m,1H, OCH₂CH═CH₂), 5.49 (s, 1H, MeOPhCH), 5.26 (dd, J=1.4, 17.3 Hz, 1H,OCH₂CH═CH₂), 5.17 (dd, J=1.4, 10.5 Hz, 1H, OCH₂CH═CH₂), 5.16 (d, J=8.1Hz, 1H, H-1), 4.65 (dd, J=3.3, 10.5 Hz, 1H, H-3), 4.37 (m, 2H,OCH₂CH═CH₂, H-6), 4.13-4.05 (3H, m, OCH₂CH═CH₂, H-4, H-6), 3.81 (s, 311,PhOCH₃), 3.75 (m, 1H, H-2), 3.48 (s, 1H, H-5), 1.05 (s, 21H,[(CH₃)₂CH]₃); ¹³C NMR (75 MHz, CDCl₃): δ=161.7, 160.1, 134.0, 130.5,127.8, 118.2, 113.6, 101.2, 97.8, 76.6, 70.6, 69.9, 69.5, 66.7, 64.2,57.6, 55.6, 18.5, 18.4, 13.1; FAB MS: m/z: calcd for C₂₅H₃₇Cl₃NO₆Si:580.1456; found: 580.1474 [M⁺-OAll].

B7) Allyl2-deoxy-2-trichloroacetamido-4,649-p-methoxybenzylidene-β-D-galactopyranoside(6). To a solution of 14′ (8.00 g, 12.5 mmol) in THF (290 mL) was addedtetrabutylammonium fluoride (1 N solution in THF, 4.91 g, 18.8 mmol,18.8 mL) and the mixture stirred at rt for 8 h. At this time a secondaddition of tetrabutylammonium fluoride (2.5 g, 9.4 mmol, 9.4 mL) wasmade and the reaction stirred for a further 12 h. The solvent wasremoved in vacuo to afford a yellow oil. Purification of this oil byflash chromatography (40%→80% EtOAc:hexanes) afforded 6 (5.14 g, 85%) asa white solid. R_(f) 0.17 (50% EtOAc:hexanes). [α]_(D) ²⁴=+0.62 (c=0.5,CH₂Cl₂); IR (thin film on NaCl): ν=3423, 1686, 1616, 1531, 1402, 1366,1303, 1249, 1170, 1097, 1060 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.43 (d,J=8.7 Hz, 2H, C₆H₄OMe), 6.89 (d, J=8.7 Hz, 2H, C₆H₄OMe), 6.87 (m, 1H,NH), 5.95-5.82 (m, 1H, OCH₂CH═CH₂), 5.54 (s, 1H, MeOPhCH), 5.29 (dd,J=1.4, 17.7 Hz, 1H, OCH₂CH═CH₂), 5.19 (dd, J=1.4, 10.5 Hz, 1H,OCH₂CH═CH₂), 4.84 (d, J=8.4 Hz, 1H, H-1), 4.44±4.32 (m, 2H, H-3, H-6),4.26-4.07 (m, 4H, OCH₂CH═CH₂, H-4, H-6), 3.81 (m, 1H, H-2), 3.81 (s, 3H,PhOCH₃), 3.53 (s, 1H, H-5), 2.71 (d, J=9.9 Hz, 1H, OH); ¹³C NMR (75 MHz,CDCl₃): δ=162.5, 160.4, 153.6, 133.7, 130.0, 127.9, 118.3, 113.8, 101.6,98.7, 75.2, 70.4, 69.4, 69.3, 67.0, 57.2, 55.7; FAB MS: m/z: calcd forC₁₉H₂₃Cl₃NO₇: 482.0540; found: 482.0531 [M+H]⁺.

C. Reaction of glucuronic Acid Monomer 5 and galactosamine Monomer 6

C1. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-β-D-galactopyranoside(4).

A mixture of donor 5 (0.50 g, 0.74 mmol) and acceptor 6 (0.30 g, 0.62mmol) was coevaporated with toluene (3×3 mL) and dried under vacuumovernight. The mixture was dissolved in CH₂Cl₂ (16 mL), and activated 4Å powdered molecular sieves were added. The reaction was stirred at rtfor 1.5 h. The reaction was then cooled to −40° C. and stirred for anadditional 30 min. Trimethylsilyl trifluoromethanesulfonate (1 M inCH₂Cl₂, 125 μL, 0.123 mmol) at −40° C. was added to the reactiondropwise. The reaction was allowed to stir an additional 30 min. It wasthen warmed to −10° C. over a period of 30 min (a carefully controlledtemperature gradient was essential to avoid formation of the inseparableortho ester) quenched with TEA, and allowed to warm to rt. The reactionwas filtered and concentrated to afford a yellow syrup. The product waspurified by flash chromatography (30% EtOAc:hexanes) to afford 4 (0.46g, 74%) as a white solid. R_(f) 0.12 (30% EtOAc:hexanes). NMR (300 MHz,CDCl₃): δ=7.87-7.82 (m, 4H, ArH), 7.48-7.39 (m, 4H, ArH), 7.35-7.26 (m,4H, Ph ArH), 6.86 (d, J=8.7 Hz, 2H, C₆H₄OMe), 6.82 (d, J=7.2 Hz, 1H,NH), 5.89-5.76 (m, 1H, OCH₂CH═CH₂), 5.45 (s, 1H, MeOPhCH), 5.52-5.39 (m,211, H-2′, H-3′), 5.22 (dd, J=1.6, 17.6 Hz, 1H, OCH₂CH═CH₂), 5.13 (dd,J=1.0, 10.4 Hz, 1H, OCH₂CH═CH₂), 5.08 (d, J=7.5 Hz, 1H, H-1′), 5.05 (d,J=8.1 Hz, 1H, H-1), 4.67 (dd, J=3.3, 10.8 Hz, 1H, H-3), 4.36-4.27 (m,4H, OCH₂CH═CH₂, H-4, H-4′ H-6), 4.10 (d, J=9.3 Hz, 1H, H-5′), 4.07-4.01(m, 2H, OCH₂CH═CH₂, H-6), 3.79 (s, 6H, CO₂CH₃, PhOCH₃), 3.77-3.68 (m,1H, H-2), 3.48 (s, 1H, H-5), 0.72 (s, 9H, (CH₃)₃CSi), −0.08 (s, 3H,CH₃Si), −0.23 (s, 3H, CH₃Si); ¹³C NMR (75 MHz, CDCl₃): δ=168.7, 165.7,165.2, 162.3, 160.0, 133.8, 133.4, 133.4, 130.5, 130.0, 129.9, 129.5,129.2, 128.5, 127.7, 118.2, 113.6, 100.7, 100.6, 97.8, 92.3, 76.4, 75.8,75.6, 73.6, 72.0, 70.9, 70.6, 69.2, 66.8, 55.6, 55.4, 52.9, 25.7, 18.1,−4.0, −4.7; FAB MS: to/z: calcd for C₄₆H₅₃Cl₃NO₁₅Si: 992.2250; found:992.2255 [M]⁺.

C2. Methyl(2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-α-D-galactopyranosidetrichloroacetimidate (7).

To a solution of 4 (2.5 g, 2.5 mmol) in dry CH₂Cl₂ (40 mL) was addedGrubbs' second generation catalyst (Scholl, M. et al. Org. Lett. 1999,1, 953-956) (0.43 g, 20 mol %) and the mixture stirred at rt for 2 h.The solvent was removed in vacuo to afford a brown oil. Purification ofthis oil by flash chromatography (15%→20% EtOAc:hexanes) affordedE/Z-prop-2-enyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsily-β-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-β-D-galactopyranoside(1.92 g, 77%) as a white solid. R_(f)(E and Z) 0.68 (60% EtOAc:hexanes).[α]_(D) ²⁵=+29.1 (c=1.0, CH₂Cl₂); IR (thin film on NaCl): ν=3308, 2954,2858, 1755, 1734, 1717, 1694, 1617, 1602, 1540, 1520, 1452, 1371, 1268,1221, 1176, 1147, 1089, 1069, 1040, 1026, 1001 cm⁻¹; ¹H NMR (300 MHz,CDCl₃): δ=7.85 (m, 3H, ArH), 7.48±7.28 (m, 10H, ArH, OCH═CHCH₃), 6.87(d, J=8.7 Hz, 2H, C₆H₄OMe), 6.82 (d, J=6.6 Hz, 1H, NH), 6.17 (m, 1H,CH═CHCH₃), 5.52-5.40 (m, 3H, MeOPhCH, H-2′, H-3′), 5.19 (d, J=8.1 Hz,1H, H-1), 5.08 (d, J=7.2 Hz, 1H, H-1′), 4.68 (dd, J=3.8, 11.0 Hz, 1H,H-3), 4.39-4.28 (m, 3H, H-4, H-4′, H-6), 4.16-4.02 (m, 2H, H-5′, H-6),3.87 (m, 1H, H-2), 3.81 (s, 3H, PhOCH₃), 3.80 (s, 3H, CO₂CH₃), 3.45 (s,1H, H-5), 1.51 (m, 3H, OCH═CHCH₃), 0.72 (s, 9H, (CH₃)₃CSi), −0.07 (s,3H, CH₃Si), −0.22 (s, 3H, CH₃Si); ¹³C NMR (75 MHz, CDCl₃): δ=168.7,165.7, 165.3, 162.4, 162.3, 160.0, 143.5, 142.1, 133.5, 133.4, 130.4,130.1, 129.9, 129.5, 129.1, 128.5, 127.7, 113.6, 105.7, 104.8, 100.8,100.6, 100.5, 98.4, 98.0, 76.5, 75.6, 75.5, 73.5, 73.4, 72.0, 70.9,69.0, 67.2, 67.1, 55.6, 55.1, 55.0, 52.9, 25.7, 18.1, 12.6, 9.7, −4.0,−4.7.

To a solution of E/Z-prop-2-enyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glueopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-β-D-galactopyranoside(6.2 g, 6.3 mmol) in dry THF (118 mL), water (24 mL) and pyridine (1.9mL) was added iodine (3.1 g) and the mixture stirred at ambienttemperature for 30 min. The solvent was removed in vacuo to afford ayellow oil. The oil was taken up in EtOAc and washed with 5% aqueousNa₂SO₃, saturated aqueous NaHCO₃, brine and dried over MgSO₄. Thesolvent was removed in vacuo to afford a pale yellow oil. Purificationof this oil by flash chromatography (40%→60% EtOAc:hexanes) afforded ananomeric mixture of methyl(2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloro-acetamido-α/β-D-galactopyranoside(4.8 g, 81%) as a pale yellow solid. R_(f) 0.28 and 0.18 (50%EtOAc:hexanes). [α]_(D) ²⁵=+79.0 (c=1.0, CH₂Cl₂); IR (thin film onNaCl): ν=3521, 2930, 1738, 1682, 1615, 1519, 1452, 1394, 1251, 1172,1093, 1069, 1031 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ=7.92-7.85 (m, 3H,ArH), 7.54-7.45 (m, 3H, ArH), 7.40-7.27 (m, 4H, ArH), 7.12 (d, J=9.0 Hz,2H, C₆H₄OMe), 6.96 (d, J=6.3 Hz, 1H, NH), 6.72 (d, J=9.0 Hz, 2H,C₆H₄OMe), 5.60 (m, 1H, H-1), 5.50 (dd, J=8.2, 8.2 Hz, 1H, H-3′), 5.42(dd, J=8.2, 8.2 Hz, 1H, H-2′), 5.24 (s, 1H, MeOPhCH), 5.21 (d, J=7.5 Hz,H-1, H-1′), 4.39-4.35 (m, 4H, H-3, H-4, H-4′), 4.23-4.02 (m, 3H, H-2,H-5′, H-6), 3.96 (s, 1H, H-5), 3.79 (s, 3H, PhOCH₃), 3.75 (s, 3H,CO₂CH₃), 3.03 (d, J=3.3 Hz, 1H, OH), 0.73 (s, 9H, (CH₃)₃CSi), −0.08 (s,3H, CH₃Si), −0.22 (s; 3H, CH₃Si); ESI MS: m/z: calcd forC₄₃H₅₀Cl₃NO₁₅Si: 954.2914; found: 954 [M−H]⁻.

To a solution of methyl(2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-fi-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-α/β-D-galactopyranoside(4.6 g, 4.8 mmol) in dry CH₂Cl₂ (190 mL) cooled to 0° C. was added1,8-diazabicyclo[5.4.0]undec-7-ene (0.29 g, 1.9 mmol, 0.29 μL) andtrichloroacetonitrile (10 g, 71 mmol, 7.2 mL) and the mixture stirredfor 15 min. The mixture was quenched with TEA and concentrated in vacuoto afford a yellow oil. Purification of this oil by flash chromatography(35% EtOAc:hexanes, +2% TEA) afforded 7 (4.7 g, 90%) as a pale yellowfoam. R_(f) 0.74, (50% EtOAc:hexanes). [α]_(D) ²⁴=+12.0 (c=0.5, CH₂Cl₂);IR (thin film on NaCl): ν=3422, 2956, 2991, 2361, 1731, 1676, 1616,1519, 1452, 1373, 1271, 1177, 1147, 1094, 1070, 1028 cm⁻¹; ¹H NMR (300MHz, CDCl₃): δ=8.69 (s, 1H, C═NH), 7.90 (m, 4H, ArH), 7.51 (m, 2H, ArH),7.42-7.26 (m, 4H, ArH), 7.00 (d, J=8.9 Hz, 211, C₆H₄OMe), 6.93 (d, J=5.4Hz, 1H, NHTCA), 6.77 (d, J=2.1 Hz, 1H, H-1), 6.68 (d, J=8.9 Hz, 2H,C₆H₄OMe), 5.52 (dd, J=8.7, 8.7 Hz, 1H, H-3′), 5.45 (dd, J=8.7, 8.7 Hz,1H, H-2′), 5.27 (d, J=7.8 Hz, 1H, H-1′), 5.17 (s, 1H, MeOPhCH), 4.62 (m,2H, H-4, H-4′), 4.49 (m, 1H, H-3), 4.31 (m, 2H, H-2, H-6), 4.18 (d,J=9.0 Hz, 1H, H-5′), 4.00 (d, J=12.6 Hz, 1H, H-6), 3.94 (s, 1H, H-5),3.75 (s, 3H, PhOCH₃), 3.74 (s, 3H, CO₂CH₃), 0.73 (s, 9H, (CH₃)₃CSi),−0.06 (s, 3H, CH₃Si), −0.19 (s, 3H, CH₃Si); ¹³C NMR (75 MHz, CDCl₃):δ=168.1, 165.9, 165.6, 162.0, 160.4, 133.9, 133.6, 130.1, 129.9, 129.4,128.7, 128.6, 127.6, 113.6, 101.1, 98.4, 95.3, 77.2, 75.5, 74.4, 71.2,70.9, 69.2, 69.0, 65.5, 55.6, 53.0, 50.5, 46.5, 25.7, −4.0, −4.8.

C3. Allyl (methyl2,3-di-O-benzoyl-β-D-glueopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-triehloroacetamido-β-D-galactopyranoside(8). To a solution of 4 (2.5 g, 2.5 mmol) in dry THF (40 mL) andpyridine (40 mL) cooled to 0° C. was added HF.pyridine (13 mL). Thereaction mixture was warmed to rt and stirred for 18 h. The mixture wasthen diluted with EtOAc and washed with 10% aqueous CuSO₄. The aqueousphase was extracted with EtOAc (3×) and the combined organics washedwith saturated aqueous NaHCO₃ and dried over MgSO₄. The solvent wasremoved in vacuo to afford a yellow oil. Purification of this oil byflash chromatography (30→60% EtOAc:hexanes) afforded 8 (1.9 g, 85%) as awhite solid. R_(f) 0.35 (60% EtOAc:hexanes). [α]_(D) ²⁵=+32.8 (c=1.0,CH₂Cl₂); IR (thin film on NaCl): ν=3422, 1731, 1616, 1519, 1452, 1369,1251, 1173, 1093, 1069 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): ν=7.93-7.87 (m,4H, ArH), 7.50-7.42 (m, 4H, ArH, C₆H₄OMe), 7.36-7.26 (m, 4H, ArH), 7.01(d, J=6.6 Hz, 1H, NH), 6.89 (d, J=8.7 Hz, 2H, C₆H₄OMe), 5.89-5.77 (m,1H, OCH₂CH═CH₂), 5.47 (m, 3H, MeOPhCH, H-2′, H-3′), 5.26-5.12 (m, 4H,OCH₂CH═CH₂, H-1, H-1′), 4.73 (dd, J=3.6, 11.4 Hz, 1H, H-3), 4.41-4.28(m, 3H, OCH₂CH═CH₂, H-4, H-6), 4.19 (m, 1H, H-4′), 4.12-4.02 (m, 3H,OCH₂CHCH₂, H-5′, H-6), 3.83 (s, 3H, PhOCH₃), 3.81 (s, 3H, CO₂CH₃), 3.72(m, 1H, H-2), 3.48 (s, 1H, H-5), 3.45 (d, J=3.3 Hz, 1H, OH); ¹³C NMR (75MHz, CDCl₃): δ=169.3, 166.6, 165.2, 162.3, 160.1, 133.8, 133.6, 133.5,130.4, 130.1, 130.0, 129.2, 129.1, 128.7, 128.6, 127.5, 118.2, 113.7,100.8, 100.7, 97.7, 76.1, 75.4, 74.3, 74.1, 71.4, 70.7, 69.3, 66.8,55.7, 53.4; ESI MS: Fez: calcd for C₄₀H₃₉Cl₃NO₁₅; 880.1; found: 880.2[M−H]⁻

C4. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-2-deoxy-2-acetamido-β-D-galactopyranoside(10). 10 was prepared using a procedure modified from Bélot et. al.(Bélot, F.; Jacquinet, J.-C. Carbohydr. Res. 2000, 326, 88-97) 4 (250mg, 0.251 mmol) was dissolved in benzene (7.80 mL). Tributylstannane(305 μL, 1.51 mmol) and 2,2′-azobisisobutyronitrile (80.0 mg) wereadded. The reaction was stirred at rt for 45 min. It was then heated to80° C. and stirred an additional 1.5 h. The reaction was cooled to rtand concentrated to afford a white solid. The product was purified byflash chromatography (50% EtOAc:hexanes) to afford allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-acetamido-β-D-galactopyranoside(190 mg, 85%) as a white solid. R_(f) 0.19 (50% EtOAc:hexanes). ¹H NMR(300 MHz, CDCl₃): δ=7.89-7.86 (m, 4H, ArH), 7.51-7.42 (m, 4H, ArH),7.37-7.31 (m, 4H, ArH), 6.88 (d, J=8.7 Hz, 21-1, C₆H₄OMe), 5.91-5.75 (m,1H, OCH₂CH═CH₂), 5.55 (dd, J=8.9, 8.9 Hz, 1H, H-3′), 5.46 (s, 1H,MeOPhCH), 5.40-5.35 (m, 2H, NH, H-2′), 5.20 (dd, J=1.4, 17.3 Hz, 1H,OCH₂CH═CH₂), 5.14-5.11 (m, 2H, OCH₂CH═CH₂, H-1), 4.97 (d, J=7.5 Hz, 1H,H-1′), 4.77 (dd, J=3.9, 11.1 Hz, 1H, H-3), 4.37-4.25 (m, 4H, OCH₂CH═CH₂,H-4, H-4′, H-6), 4.10 (d, J=9.6 Hz, 1H, H-5′), 4.10-3.98 (m, 2H,OCH₂CH═CH₂, H-6), 3.81 (s, 3H, CO₂CH₃), 3.78 (s, 3H, PhOCH₃), 3.47 (s,1H, H-5), 3.34-3.26 (m, 1H, H-2), 1.53 (s, 3H, HNC(O)CH₃), 0.72 (s, 9H,(CH₃)₃CSi), −0.07 (s, 3H, CH₃Si), −0.23 (s, 3H, CH₃Si); ¹³C NMR (75 MHz,CDCl₃): δ=171.4, 168.7, 165.8, 165.0, 160.0, 134.1, 133.5, 133.4, 130.7,129.9, 129.8, 129.6, 129.5, 128.6, 128.5, 127.8, 118.0, 113.6, 101.6,100.8, 98.0, 76.3, 76.1, 75.6, 72.4, 70.9, 70.4, 69.4, 66.7, 55.6, 55.1,52.9, 25.8, 23.6, 18.1, −4.0, −4.7; ESI MS: [M+Na]⁺calcd forC₄₆H₅₇NNaO₁₅Si: 914.3, found 914.4.

Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glueopyranosyluronate)-(1→3)-4,6-O-p-methoxybenzylidene-2-deoxy-2-acetamido-β-D-galactopyranoside(190 mg, 0.213 mmol) was dissolved in CH₂Cl₂ (2.40 mL) and H₂O (0.560mL). 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (73.0 mg, 0.320 mmol) wasadded. The reaction was stirred at rt for 3 h, quenched with MeOH, andconcentrated to yield a red solid. The product was purified on SephadexLH-20 (50% CH₂Cl₂:MeOH), followed by silica gel chromatography (100%EtOAc), to afford an orange solid containing the desired diol 10 (102mg, 62%). R_(f) 0.23 (100% EtOAc). ¹H NMR (300 MHz, CD₃OD): δ=7.90-7.86(m, 4H, ArH), 7.49-7.44 (m, 2H, ArH), 7.37-7.29 (m, 4H, ArH), 5.83-5.74(m, 1H, OCH₂CH═CH₂), 5.61 (dd, J=9.0, 8.7 Hz, 1H, H-3′), 5.35 (dd,J=8.1, 9.0 Hz, 1H, H-2′), 5.18 (dd, J=1.7, 17.6 Hz, 1H, OCH₂CH═CH₂),5.10 (d, J=9.9 Hz, 1H, OCH₂CH═CH₂), 4.97 (d, J=7.5 Hz, 1H, H-1′), 4.90(d, J=7.5 Hz, 1H, H-1), 4.552 (m, 1H, NH), 4.29-4.17 (m, 3H, H-1, H-4,H-6), 4.03-3.93 (m, 4H, OCH₂CH═CH₂, H-3, H-4′, H-6), 3.89-3.86 (m, 2H,OCH₂CH═CH₂, H-5, H-5′), 3.65 (m, 1H, H-2), 3.78 (s, 3H, CO₂CH₃), 1.26(s, 3H, HNC(O)CH₃), 0.73 (s, 9H, (CH₃)₃CSi), −0.07 (s, 3H, CH₃Si), −0.20(s, 3H, CH₃Si); ESI MS: m/z: calcd for C₃₉H₅₂NO₁₄Si 774.9; found 774.2[M+H]⁺.

C5. Allyl (sodium β-D-glucopyranosyluronate)-(1→3)-4,6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranoside (1). The crude diol10 (102 mg, 0.132 mmol) was dissolved in DMF (5 mL). SO₃.TMA (0.550 g,3.96 mmol) was added. The reaction was stirred at 50° C. overnight. Itwas cooled to rt, quenched with MeOH, and concentrated to afford ayellow solid. The product was purified on Sephadex LH-20 (50%CH₂Cl₂:MeOH), followed by silica gel chromatography (10%→20%MeOH:CH₂Cl₂), to afford allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranoside (115 mg, 93%) as awhite solid. R_(f) 0.125 (15% MeOH:CH₂Cl₂). ¹H NMR (300 MHz, CD₃OD):7.88-7.85 (m, 4H, ArH), 7.54-7.47 (m, 2H, ArH), 7.38-7.32 (m, 411, ArH),5.86-5.73 (m, 1H, OCH₂CH═CH₂), 5.67 (dd, J=9.3, 9.3 Hz, 1H, H-3′), 5.48(dd, J=8.1, 9.2 Hz, 1H, H-2′), 5.18 (dd, J=1.7, 17.6 Hz, 1H,OCH₂CH═CH₂), 5.11 (d, J=7.5 Hz, 1H, H-1′), 5.05 (dd, J=1.8, 10.5 Hz, 1H,OCH₂CH═CH₂), 4.44-4.35 (m, 3H, H-1, H-4, H-6), 4.30-4.22 (m, 4H,OCH₂CH═CH₂, H-3, H-4′, H-6), 4.09-3.98 (m, 2H, OCH₂CH═CH₂, H-5),3.95-3.91 (m, 2H, H-2, H-5′), 3.86 (s, 3H, CO₂CH₃), 1.30 (s, 3H,HNC(O)CH₃), 0.74 (s, 9H, (CH₃)₃CSi), −0.02 (s, 3H, CH₃Si), −0.18 (s, 3H,CH₃Si); ¹³C NMR (75 MHz, CD₃OD): δ=172.3, 170.0, 166.2, 165.8, 134.1,133.5, 133.4, 130.0, 129.5, 129.4, 129.0, 128.3, 128.2, 115.9, 102.5,100.8, 79.2, 76.3, 75.9, 75.4, 72.8, 72.6, 70.9, 69.7, 67.6, 54.2, 52.6,25.0, 21.4, 17.6, −4.9, −5.6; FAB MS: In/z: calcd for C₃₈H₄₉NNa₃O₂₀S₂Si:1000.175; found: 1000.175 [M+Na]⁺.

Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-4,6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranoside (115 mg, 0.123 mmol)was dissolved in pyridine (1.7 mL) and THF (1.7 mL). The reaction wascooled to 0° C., HF.pyridine (0.60 mL) was added, and it slowly warmedto rt overnight. After 12 h, the mixture was flowed through a SephadexLH-20 column (50% CH₂Cl₂:MeOH) and the concentrated residue was purifiedby silica gel chromatography (10%→20% MeOH:CH₂Cl₂) to afford a whitesolid (90.0 mg). R_(f) 0.50 (EtOAc:pyr:H₂O:AcOH, 8:5:3:1).

The crude alcohol (90 mg, 0.11 mmol) was dissolved in THF (1.8 mL) andH₂O (1.8 mL) and to this was added 2 M NaOH (0.72 mL). After 12 h at rt,the reaction was neutralized with Amberlyst IR-120 resin, filtered, andlyophilized to afford an orange solid. The product was purified onSephadex G-10 (100% H₂O) and Sephadex SP C25 (100% H₂O) and lyophilizedto afford 1 (45 mg, 55%, 2 steps) as a white solid. R_(f) 0.12(EtOAc:pyr:H₂O:AcOH, 8:5:3:1). ¹H NMR (300 MHz, D₂O): δ=5.79-5.66 (m,1H, OCH₂CH═CH₂), 5.17-5.07 (m, 2H, OCH₂CH═CH₂), 4.42-4.39 (m, 1H), 4.31(d, J=7.8 Hz, 1H, H-1′), 4.16-4.10 (m, 2H), 4.05-3.98 (m, 3H), 3.90-3.87(m, 3H), 3.53 (dd, J=9.0, 9.0 Hz, 1H), 3.36-3.29 (m, 2H), 3.21-3.16 (m,1H), 1.84 (s, 3H, HNC(O)CH₃); ¹³C NMR (75 MHz, D₂O): δ=118.7, 103.4,100.0, 175.6, 174.8, 133.2, 76.4, 75.2, 75.1, 72.6, 72.4, 71.9, 70.8,68.0, 51.8, 22.5; FAB MS: m/z: calcd for C₁₇H₂₄NNa₂O₁₈S₂: 640.0230;found: 640.0202 [M−Na]⁻.

C6. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glueopyranosyluronate)-(1→6)-(4,6-O-p-methoxybenzylidene-2-deoxy-2-triehloroacetamide-β-D-galactopyranosyl)-(1→4)-(methyl2,3-di-O-benzoyl-p-D-glucopyranosyluronate)-(1→6)-4,6-O-p-methoxybenzylidene-2-deoxy-2-trichloroacetamido-β-D-galactopyranoside(15′). 7 (0.20 g, 0.182 mmol) and 8 (0.13 g, 0.15 mmol) were combinedand coevaporated with toluene (3×) and put under high vacuum overnightto dry. The mixture was dissolved in CH₂Cl₂ (3.0 mL) and 4 Å powderedmolecular sieves added. The mixture was stirred for 1 h at rt and thencooled to −15° C. Trimethylsilyl trifluoromethanesulfonate (0.5 Nsolution in CH₂Cl₂, 0.0068 g, 0.031 mmol, 61 μL) was added and thereaction was stirred at −15° C. for 30 min and then quenched with TEA.The mixture was filtered and concentrated to afford a yellow oil.Purification of this oil by flash chromatography (30→40% EtOAc:hexanescontaining 0.1% TEA) afforded 15′ (85 mg, 31%) as a white solid. R_(f)0.43 (60% EtOAc:hexanes). [α]_(D) ²⁵=+13.4 (c=0.5, CH₂Cl₂); IR (thinfilm on NaCl): ν=3424, 2956, 2361, 1732, 1638, 1519, 1452, 1368, 1251,1173, 1093, 1173, 1093, 1070, 1028; ¹H NMR (600 MHz, CDCl₃): δ=7.88-7.80(m, 8H, ArH), 7.49-7.45 (m, 4H, ArH), 7.38-7.28 (m, 81-1, ArH),7.22-7.20 (m, 2H, ArH), 7.06 (d, J=8.4 Hz, 2H, C₆H₄OMe), 6.93 (d, J=8.4Hz, 2H, C₆H₄OMe), 6.85 (d, J=6.6 Hz, 1H, NH″), 6.74 (d, J=8.4 Hz, 2H,Ph), 6.66 (d, J=7.2 Hz, 1H, NH), 5.87-5.81 (m, 1H, OCH₂CH═CH₂), 5.58(dd, J=7.8, 7.8 Hz, 1H, H-3′), 5.49 (s, 1H, MeOPhCH), 5.44 (dd, J=8.7,8.7 Hz, 1H, 1H, H-3″), 5.35 (m, 2H, H-2′, H-2′″), 5.23 (d, J=18.0 Hz,1H, OCH₂CH═CH₂), 5.20 (s, 1H, MeOPhCH), 5.15 (m, 2H, OCH₂CH═CH₂, H-1′),5.11 (d, J=7.8 Hz, 1H, H-1″), 5.03 (d, J=7.2 Hz, 1H, H-1′″), 5.00 (d,J=8.4 Hz, 1H, H-1), 4.68 (dd, J=3.6, 10.8 Hz, 1H, H-3″), 4.58 (dd,J=9.0, 9.0 Hz, 1H, H-4′), 4.39-4.30 (m, 5H, OCH₂CH═CH₂, H-3, H-4″,H-4′″, H-6″), 4.14 (m, 2H, H-4, H-5′), 4.06 (m, 3H, OCH₂CH═CH₂, H-5′″,H-6″), 3.83 (s, 3H, PhOCH₃), 3.81-3.68 (m, 4H, H-2, H-2″, H-6, H-6),3.80 (s, 3H, PhOCH₃), 3.80 (s, 3H, CO₂CH₃), 3.79 (s, 3H, CO₂CH₃), 3.48(s, H-1, H-5″), 3.10 (s, 1H, H-5), 0.72 (s, 9H, (CH₃)₃CSi), −0.09 (s,31-1, CH₃Si), −0.24 (s, 3H, CH₃Si); ¹³C NMR (75 MHz, CDCl₃): Et=168.8,168.4, 165.7, 165.4, 165.2, 165.1, 162.2, 161.9, 160.0, 159.8, 133.8,133.4, 133.3, 133.1, 130.5, 130.4, 130.2, 130.1, 130.0, 129.9, 129.6,129.5, 129.2, 129.1, 128.6, 128.5, 128.4, 127.9, 127.8, 118.2, 113.7,113.4, 100.8, 100.5, 100.4, 100.2, 98.6, 97.7, 77.4, 76.4, 75.9, 75.8,75.3, 75.0, 74.2, 74.1, 73.5, 73.4, 72.1, 71.9, 70.8, 70.6, 69.3, 68.4,66.9, 55.7, 55.6, 54.8, 53.5, 52.8, 25.7, 18.1, −4.1, −4.8. ESI MS: m/z:calcd for C₈₃H₈₉Cl₆N₂O₂9Si: 1819.4; found 1820.4 [M+H]⁺.

C7. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-(4,6-O-p-methoxybenzylidene-2-deoxy-2-acetamido-β-D-galactopyranosyl)-(1→4)-(methyl2,3-di-O-benzoyl-β-D-glueopyranosyluronate)-(1→3)-4,6-O-p-met-hoxybenzylidene-2-deoxy-2-acetamido-β-D-galactopyranoside(16′). 15′ (50 mg, 0.027 mmol) was dissolved in benzene (0.88 mL) andN,N-dimethylacetamide (0.22 mL) and to this were added tributylstamiane(0.10 mL, 0.49 mmol) and 2,2′-azobisisobutyronitrile (2.0 mg). Thereaction was stirred at rt for 30 min and then was heated at 80° C. for5 h. It was cooled to rt, concentrated to afford a yellow-white solid,and purified by silica gel chromatography (80%→100% EtOAc:hexanes) toyield 16′ as a white solid (37 mg, 85%). R_(f) 0.69 (100% EtOAc). ¹H NMR(300 MHz, CDCl₃): δ=7.95-7.84 (m, 8H, ArH), 7.52-7.43 (m, 6H, ArH),7.38-7.27 (m, 8H, ArH), 7.21 (d, J=9.0 Hz, 2H, C₆H₄OMe), 6.86 (d, J=8.7Hz, 2H, C₆H₄OMe), 6.80 (d, J=9.0 Hz, 2H, Ph C₆H₄OMe), 5.89-5.76 (m, 1H,OCH₂CH═CH₂), 5.61 (dd, J=7.2, 8.1 Hz, 1H, H-3′), 5.51 (s, 1H, MeOPhCH),5.44 (dd, J=8.7, 9.0 Hz, 1H, H-3′″), 5.42 (d, J=6.6 Hz, 1H, NH″), 5.31(dd, J=6.6, 7.2 Hz, 1H, H-2′), 5.28 (dd, J=7.2, 8.7 Hz, 1H, H-2″), 5.20(dd, J=0.9, 17.3 Hz, 1H, OCH₂CH═CH₂), 5.18 (s, 1H, MeOPhCH), 5.13 (d,J=11.4 Hz, 1H, OCH₂CH═CH₂), 5.11 (d, J=8.1 Hz, 1H, H-1′), 5.05 (d, J=7.2Hz, 1H, H-1″), 4.98 (d, J=6.6 Hz, H, NH), 4.89 (d, J=7.5 Hz, 1H, H-1),4.86 (d, J=9.0 Hz, 1H, H-1′″), 4.75 (dd, J=3.3, 10.8 Hz, 1H, H-3″), 4.51(dd, J=8.1, 9.3 Hz, 1H, H-4′), 4.37-4.25 (m, 5H, OCH₂CH═CH₂, H-3, H-4″,H-4′″, H-6″), 4.16 (d, J=9.3 Hz, 1H, H-5′″), 4.06-3.98 (m, 4H,OCH₂CH═CH₂, H-4, H-5′, H-6″), 3.77-3.73 (m, 1H, H-6), 3.80 (s, 3H,PhOCH₃), 3.79 (s, 3H, PhOCH₃), 3.73 (s, 3H, CO₂CH₃), 3.70 (s, 3H,CO₂CH₃), 3.56-3.52 (m, 1H, H-6), 3.46 (s, 1H, H-5″), 3.35-3.26 (m, 2H,H-2, H-2″), 2.84 (s, 1H, H-5), 1.54 (s, 3H, HNC(O)CH₃), 1.50 (s, 3H,HNC(O)CH₃), 0.70 (s, 9H, (CH₃)₃CSi), −0.10 (s, 3H, CH₃Si), −0.25 (s, 3H,CH₃Si). ESI MS: m/z: calcd for C₈₃H₉₄N₂O₂₉Si: 1647.2; found 1648.0[M+Cl]⁻.

C8. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glueopyranosyluronate)-(1→3)-(2-deoxy-2-acetamido-β-D-gal-actopyranosyl)-(1→4)-(methyl2,3-di-O-benzoyl-β-D-glueopyranosyluronate)-(1→3)-2-deoxy-2-acetamido-βD-galactopyranoside(9). 16′ (13 mg, 0.0083 mmol) was dissolved in CH₂Cl₂ (200 and H₂O (24μL) and the reaction was covered with aluminum foil and stirred in thedark. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (6.0 mg, 0.025 mmol) wasadded and the reaction stirred for 2 h at rt. The reaction was quenchedwith MeOH and concentrated to afford a red solid. The crude product wassubjected to Sephadex LH-20 (50% CH₂Cl₂:MeOH) to afford 9 as a yellowsolid (8.5 mg, 75%). R_(f) 0.2 (100% EtOAc). ¹H NMR (300 MHz, CD₃OD):δ=7.85-7.76 (m, 8H, ArH), 7.47-7.42 (m, 4H, ArH), 7.36-7.27 (m, 8H,ArH), 5.79-5.66 (m, 1H, OCH₂CH═CH₂), 5.52 (dd, J=8.4, 8.4 Hz, 1H, H-3′),5.51 (dd, J=8.4, 9.9 Hz, 1H, H-3″), 5.27-5.19 (m, 3H), 5.12 (dd, J=1.6Hz, 17.3 Hz, 1H, OCH₂CH═CH₂), 5.00-4.96 (m, 4H), 4.43-4.42 (m, 1H),4.32-4.26 (m, 2H), 4.20-4.10 (m, 5H), 4.00 (d, J=2.4 Hz, 1H), 3.96-3.88(m, 3H), 3.70 (s, 3H, CO₂CH₃), 3.69 (s, 3H, CO₂CH₃), 3.41-3.35 (m, 2H),3.17-3.10 (m, 3H), 3.04-3.00 (m, 1H), 1.20 (s, 3H, HNC(O)CH₃), 1.18 (s,3H, HNC(O)CH₃), 0.66 (s, 9H, (CH₃)₃CSi), −0.10 (s, 3H, CH₃Si), −0.26 (s,3H, CH₃Si). ESI MS: m/z: calcd for C₆₇H₈₂N₂NaO₂₇Si: 1398.4; found 1397.6[M+Na]⁺.

C9. Allyl (methyl2,3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-β-D-glueopyranosyluronate)-(1→3)-(4,6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranosyl)-(1→4)-(methyl2,3-di-O-benzoyl-β-D-glueopyranosyluronate)-(1→3)-4,6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranoside (17′). 9 (13 mg,0.0095 mmol) was dissolved in DMF (315 μL) and to this was added SO₃.TMA(50 mg, 0.36 mmol) and the reaction stirred at 50° C. for 2 d. It wasquenched with MeOH, concentrated to afford a yellow solid, and purifiedon Sephadex LH-20 (50% CH₂Cl₂:MeOH) and Sephadex SP C25 (50% H₂O:MeOH)to afford 17′ as a white solid (11 mg, 67%). R_(f) 0.29(EtOAc:pyr:H₂O:AcOH, 8:5:3:1). ¹H NMR (300 MHz, CD₃OD): δ=7.92-7.81 (m,8H, ArH), 7.55-7.45 (m, 4H, ArH), 7.43-7.33 (m, 8H, ArH), 5.87-5.73 (m,1H, OCH₂CH═CH₂), 5.67 (dd, J=9.0, 9.0 Hz, 1H, H-3′), 5.61 (dd, J=9.3,9.3 Hz, 1H, H-3′″), 5.42-5.32 (m, 3H), 5.19 (dd, J=1.6, 17.3 Hz, 1H,OCH₂CH═CH₂), 4.93-4.79 (m, 4H, H-4, H-4″), 4.54-4.52 (m, 1H), 4.49 (dd,J=9.0, 9.6 Hz, 1H, H-4′), 4.40-4.33 (m, 5H), 4.28-4.22 (m, 3H), 4.18 (d,J=9.3 Hz, 1H, H-5′″), 4.08-3.98 (m, 4H), 3.90-3.89 (m, 1H), 3.87 (s, 3H,CO₂CH₃), 3.86-3.85 (m, 2H), 3.83 (s, 3H, CO₂CH₃), 1.20 (s, 3H,HNC(O)CH₃), 1.18 (s, 3H, HNC(O)CH₃), 0.73 (s, 9H, (CH₃)₃CSi), −0.03 (s,3H, CH₃Si), −0.19 (s, 3H, CH₃Si). ESI MS: m/z: calcd forC₆₇H₇₈N₂Na₃O₃₉S₄Si: 1760.6; found 1759.8 [M−Na]⁻.

C10. Allyl (sodium β-D-glueopyranosyluronate)-(1→3)-(4,-6-di-O-sodiumsulfonato-2-deoxy-2-acetamido-β-D-galactopyranosyl)-(1→4)-(sodiumβ-D-glucopyranosyluronate)-(1sulfonato-2-deoxy-2-acetamido-β-D-galactopyranoside (2). 17′ (11 mg,0.0062 mmol) was dissolved in pyridine (150 μL), THF (150 μL), and H₂O(35 μl). The reaction was cooled to 0° C. and to this was addedHF.pyridine (41 μL). It stirred at 0° C. for 1 h and at rt overnight,and following this, was loaded onto a Sephadex LH-20 (50% CH₂Cl₂:MeOH)column. The product was concentrated, taken up in H₂O, and lyophilizedto afford a white solid (4.9 mg) that was immediately used in the nextreaction.

The alcohol was deprotected in a manner similar to a procedure fromLucas et. al. (Lucas, H.; Basten, J. E. M.; van Dinther, T. G.;Meuleman, D. G.; van Aelst, S. F.; van Boeckel, C. A. A. Tetrahedron,1990, 46, 8207-8228) The alcohol (4.9 mg, 0.0033 mmol) was dissolved inTHF (190 μL) and H₂O (94 μL) and cooled to 0° C. To this were added 1 Maq. LiOH (75 mL) and 30% H₂O₂ (38 μL). The reaction stirred at 0° C. for1 h and at rt for 12 h. At this time, 4 M NaOH (56 μL) and MeOH (280 μL)were added and the reaction stirred for another 12 h. It was thenneutralized with Amberlyst IR-120 resin, filtered, and lyophilized toafford an orange solid. The product was purified by Sephadex G-25 UF(0.9% NaCl in H₂O) and desalted with Sephadex G-25 UF (100% H₂O) toafford 2 as a white solid upon lyophilization (1.9 mg, 25% from 17′). ¹HNMR (600 MHz, D₂O): δ=5.93-5.89 (m, 1H, OCH₂CH═CH₂), 5.32 (d, J=17.4 Hz,1H, OCH₂CH═CH₂), 5.13 (d, J=10.2 Hz, 1H, OCH₂CH═CH₂), 4.80-4.73 (m, 2H,H-4, H-4″), 4.62-4.55 (m, 2H), 4.50-4.46 (m, 2H), 4.34 (dd, J=4.8, 12.6Hz, 1H, OCH₂CH═CH₂), 4.29 (d, J=10.2 Hz, 1H), 4.24-4.18 (m, 2H), 4.13(d, J=7.8 Hz, 1H), 4.06 (d, J=10.8 Hz, 4H), 3.83-3.75 (m, 2H), 3.70-3.63(m, 3H), 3.60 (dd, J=7.8, 9.6 Hz, 1H, H-3′), 3.57-3.51 (m, 2H), 3.47(dd, J=9.0, 9.6 Hz, 1H, H-3′), 3.41 (dd, J=8.4, 8.4 Hz, 1H, H-2′), 3.34(dd, J=8.4, 8.4 Hz, 1H, H-2′″), 2.04 (s, 3H, HNC(O)CH₃), 2.01 (s, 3H,HNC(O)CH₃). ESI MS: m/z: calcd for C₃₁H₄₂N₂Na₅O₃₅S₄: 1245.9; found1245.0 [M−Na]⁻.

C11. Allyl (sodiumβ-D-glucopyranosyluronate)-(1→3)-(2-deoxy-2-acetamido-β-D-galactopyranosyl)-(1→4)-(sodiumβ-D-glucopyranosyluronate)-(1→3)-2-deoxy-2-acetamido-β-D-galactopyranoside(3). 9 (8.5 mg, 0.0062 mmol) was dissolved in pyridine (110 and THF (110μL). The reaction was cooled to 0° C. and to this was added HFpyridine(30 mL). The reaction stirred at 0° C. for 1 h and at rt overnight.Following this, the mixture was loaded onto a Sephadex LH-20 (50%CH₂Cl₂:MeOH) column and the product was a yellow solid (5.3 mg) that wasimmediately used in the next reaction. The alcohol (5.3 mg, 0.0042 mmol)was dissolved in THF (120 μL) and H₂O (60 μL) and cooled to 0° C. Tothis were added 1 M aq. LiOH (47 μL) and 30% H₂O₂ (23 μL). The reactionstirred at 0° C. for 1 h and at rt for 12 h. At this time, 4 M NaOH (35μL) and MeOH (173 μL) were added and the reaction stirred for another 12h. It was neutralized with Amberlyst IR-120 resin, filtered, andlyophilized to afford an orange solid. The product was purified bySephadex G-250F (100% H₂O) and lyophilized to afford 3 as a white solid(2.6 mg, 52% from 9). ¹H NMR (600 MHz, D₂O): δ=5.91-5.84 (m, 1H,OCH₂CH═CH₂), 5.28 (d, J=17.4 Hz, 1H, OCH₂CH═CH₂), 5.23 (d, J=10.2 Hz,1H, OCH₂CH═CH₂), 4.51-4.45 (m, 41-1), 4.31 (dd, J=4.8, 12.9 Hz, 1H,OCH₂CH═CH₂), 4.16-4.09 (m, 3H), 4.01-3.96 (m, 2H), 3.78-3.71 (m, 5H),3.67-3.64 (m, 5H), 3.55 (dd, J=9.0, 9.0 Hz, 1H, H-3′″), 3.48-3.42 (m,3H), 3.34 (dd, J=8.4, 9.0 Hz, 1H, H-2′), 3.29 (dd, J=7.2, 8.4 Hz, 1H,H-2′″), 1.99 (s, 3H, HNC(O)CH₃), 1.98 (s, 3H, HNC(O)CH₃). ESI MS: m/z:calcd for C₃₁H₄₇N₂O₂₃: 815.7; found 815.4 [M−H]⁻.

Example 2

The following tetrasaccharide were synthesized using similar proceduresas described in example 1:

Example 3 Effect on Hippocampal Neuronal Growth

Hippocampal Neuronal Cultures. Hippocampal neuronal cultures wereprepared using a modified version of the Goslin and Banker protocol(Goslin, K.; Banker, G. In Culturing Nerve Cells; Banker, G.; Goslin,K.; Eds.; MIT Press: Cambridge, Mass., 1991; pp 251-281). Embryos at theE18 stage were obtained from timed-pregnant Sprague-Dawley rats, and thehippocampus from each embryo was dissected. All the hippocampi from onepreparation were transferred to a 15 mL conical tube containing 4.5 mLof ice-cold Calcium and Magnesium Free-Hank's Balanced Salt Solution(CMF-HBSS) (GIBCO). Trypsin (2.5%, no EDTA; GIBCO) was added to 5 mL,and the tissue was digested for 15 mM at 37° C. The trypsin solution wasremoved and the tissue rinsed with 5 mL of CMF-HBSS three times. Thetissue was then dissociated in 1 mL of CMF-HBSS by passing through aP1000 pipet tip twenty times. The cells were counted with ahemacytometer and plated on glass coverslips at 80 cells/mm² andcultivated in minimal Eagle's Medium (MEM) (GIBCO) supplemented with theN2 mixture (GIBCO) and 0.1 mM pyruvate. The cultures were maintained in5% CO₂ at 37° C. Glass coverslips were coated as described by Clementet. al. (Clement, A. M.; Nadanaka, S.; Masayama, K.; Mandl, C.;Sugahara, K.; Faissner, A. J. Biol. Chem. 1998, 273, 28444-28453)Briefly, coverslips were precoated with 0.015 mg/mL poly-DL-ornithine(SIGMA) for 1 h at 37° C./5% CO₂, washed three times with doubledistilled H₂O, and coated with 0.5 mg/mL of compounds 1-3 in PBSovernight at 37° C./5% CO₂. The coverslips were then washed three timeswith PBS and flooded with MEM+N2 media.

Immunocytochemistry of Hippocampal Neuronal Cultures. After 48 h inculture, hippocampal neurons on coverslips were used for immunostaining.Cells were rinsed one time with PBS, fixed in 4% paraformaldehyde for 20min at rt, washed twice with PBS, permeablized in 0.3% Triton X-100 for5 min at rt, and washed twice with PBS. Non-specific binding was blockedwith 3% BSA for 1 h at rt. The blocking solution was rinsed off one timewith PBS. Cells were then incubated with anti-tau antibodies (rabbitpolyclonal, 1:600; SIGMA) in 3% BSA for 2 h at rt. Excess antibody wasrinsed away 5 times with PBS. Fluorophore-conjugated secondaryantibodies were purchased from Molecular Probes and added for 1 h at 37°C. in 3% BSA. The secondary antibody used was anti-rabbit IgG AlexaFluor488 (1:600). Excess secondary antibody was washed off 5 times with PBS.The coverslips were mounted onto glass slides using Vectashield mountingmedium (Vector Labs) and sealed with clear nail polish. Cells were thensubjected to confocal laser microscopy.

Confocal Laser Microscopy. All cells were imaged on a Zeiss Axiovert100M inverted confocal laser microscope in the Biological Imaging Centerin the Beckman Institute at Caltech. The images were captured with LSMPascal software using a 40× plan-neofluar oil objective. All cells wereexcited with 488 nm light.

Morphometric Analysis. For quantitative analysis, 50 cells were analyzedper coverslip. Only cells with neurites longer than one cell bodydiameter were counted. The length of the longest neurite from stainedcells was measured using NIH Image 1.52 software. The mean neuritelengths were compared among the different substrate conditions by theANOVA test using the statistical analysis program StatView (SASInstitute Inc., Cary, N.C.).

Since modifications will be apparent to those of skill in the art, it isintended that the invention be limited only by the scope of the appendedclaims.

1.-57. (canceled)
 58. A method of modulating neuronal growth, saidmethod comprising contacting a cell with an effective amount of acompound of the following formula:

or a pharmaceutically acceptable derivative thereof, wherein each of R1,R2, R3, and R4 are independently selected as follows: (i) R1 and R2 areeach independently selected from sulfate, phosphate and carboxylate; andR3 and R4 are hydrogen; (ii) R3 and R4 are each independently selectedfrom sulfate, phosphate, carboxylate; and R1 and R2 are hydrogen; (iii)R1, R2 and R3 are each independently selected from sulfate, phosphate,carboxylate; and R4 is hydrogen; (iv) R1, R2, and R4 are eachindependently selected from sulfate, phosphate, carboxylate; and R3 ishydrogen; (v) R2 and R4 are each independently selected from sulfate,phosphate, carboxylate; and R1 and R3 are hydrogen; (vi) R1 is selectedfrom sulfate, phosphate, carboxylate; and R2, R3, and R4 are hydrogen;(vii) R2 is selected from sulfate, phosphate, carboxylate; and R1, R3,and R4 are hydrogen; (viii) R3 is selected from sulfate, phosphate,carboxylate; and R1, R2, and R4 are hydrogen; or (ix) R4 is selectedfrom sulfate, phosphate, carboxylate; and R1, R2, and R3 are hydrogen;with the provisos that (a) when R1 is sulfate, then R2 is other than H;(b) when R2 is sulfate then R1 is other than H; and R5 is selected fromoptionally substituted alkyl and optionally substituted alkenyl; and nis 0-100.
 59. The method of claim 58, wherein the method results inpromotion of neurite outgrowth.
 60. The method of claim 58, wherein themethod results in inhibition of neurite outgrowth.
 61. The method ofclaim 58, wherein the cell is from a neuronal tissue.
 62. The method ofclaim 58, wherein the cell is a cell from a brain, a central nervoussystem, or a peripheral nervous system.
 63. The method of claim 58,wherein the method results in inhibiting regeneration of an injurednerve.
 64. The method of claim 58, wherein the method results inpromoting regeneration of an injured nerve.
 65. The method of claim 64,wherein the cultured neuronal cell is a hippocampal neuron, adopaminergic neuron, or a dorsal root ganglion neuron.
 66. A method ofinducing growth of a differentiated neural stem cell comprisingcontacting a neural stern cell with an effective amount of a compound ofthe following formula:

or a pharmaceutically acceptable derivative thereof, wherein each of R1,R2, R3, and R4 are independently selected as follows: (i) R1 and R2 areeach independently selected from sulfate, phosphate and carboxylate; andR3 and R4 are hydrogen; (ii) R3 and R4 are each independently selectedfrom sulfate, phosphate, carboxylate; and R1 and R2 are hydrogen; (iii)R1, R2 and R3 are each independently selected from sulfate, phosphate,carboxylate; and R4 is hydrogen; (iv) R1, R2, and R4 are eachindependently selected from sulfate, phosphate, carboxylate; and R3 ishydrogen; (v) R2 and R4 are each independently selected from sulfate,phosphate, carboxylate; and R1 and R3 are hydrogen; (vi) R1 is selectedfrom sulfate, phosphate, carboxylate; and R2, R3, and R4 are hydrogen;(vii) R2 is selected from sulfate, phosphate, carboxylate; and R1, R3,and R4 are hydrogen; (viii) R3 is selected from sulfate, phosphate,carboxylate; and R1, R2, and R4 are hydrogen; or (ix) R4 is selectedfrom sulfate, phosphate, carboxylate; and R1, R2, and R3 are hydrogen;with the provisos that (a) when R1 is sulfate, then R2 is other than H;(b) when R2 is sulfate then R1 is other than H; and R5 is selected fromoptionally substituted alkyl and optionally substituted alkenyl; and nis 0-100.
 67. A method of treating a symptom associated with aneurological disorder comprising administering to a subject in needthereof an effective amount of a compound of the following formula:

or a pharmaceutically acceptable derivative thereof, wherein each of R1,R2, R3, and R4 are independently selected as follows: (i) R1 and R2 areeach independently selected from sulfate, phosphate and carboxylate; andR3 and R4 are hydrogen; (ii) R3 and R4 are each independently selectedfrom sulfate, phosphate, carboxylate; and R1 and R2 are hydrogen; (iii)R1, R2 and R3 are each independently selected from sulfate, phosphate,carboxylate; and R4 is hydrogen; (iv) R1, R2, and R4 are eachindependently selected from sulfate, phosphate, carboxylate; and R3 ishydrogen; (v) R2 and R4 are each independently selected from sulfate,phosphate, carboxylate; and 121 and R3 are hydrogen; (vi) R1 is selectedfrom sulfate, phosphate, carboxylate; and R2, R3, and R4 are hydrogen;(vii) R2 is selected from sulfate, phosphate, carboxylate; and R1, R3,and R4 are hydrogen; (viii) R3 is selected from sulfate, phosphate,carboxylate; and R1, R2, and R4 are hydrogen; or (ix) R4 is selectedfrom sulfate, phosphate, carboxylate; and R1, R2, and R3 are hydrogen;with the provisos that (a) when R1 is sulfate, then R2 is other than H;(b) when R2 is sulfate then R1 is other than H; and R5 is selected fromoptionally substituted alkyl and optionally substituted alkenyl; and nis 0-100.
 68. The method of claim 67, wherein the disorder is a CNSlesion, gliosis, Parkinson's Disease, Alzheimer's Disease, or neuronaldegeneration.
 69. The method of claim 67, wherein the disorder is spinalcord trauma.
 70. A method of modulating the activity of a growth factorcomprising contacting a cell with an effective amount a compoundcomprising:

or a pharmaceutically acceptable derivative thereof, wherein each of R1,R2, R3, and R4 are independently selected as follows: (i) R1 and R2 areeach independently selected from sulfate, phosphate and carboxylate; andR3 and R4 are hydrogen; (ii) R3 and R4 are each independently selectedfrom sulfate, phosphate, carboxylate; and R1 and R2 are hydrogen; (iii)R1, R2 and R3 are each independently selected from sulfate, phosphate,carboxylate; and R4 is hydrogen; (iv) R1, R2, and R4 are eachindependently selected from sulfate, phosphate, carboxylate; and R3 ishydrogen; (v) R2 and R4 are each independently selected from sulfate,phosphate, carboxylate; and R1 and R3 are hydrogen; (vi) R1 is selectedfrom sulfate, phosphate, carboxylate; and R2, R3, and R4 are hydrogen;(vii) R2 is selected from sulfate, phosphate, carboxylate; and R1, R3,and R4 are hydrogen; (viii) R3 is selected from sulfate, phosphate,carboxylate; and R1, R2, and R4 are hydrogen; or (ix) R4 is selectedfrom sulfate, phosphate, carboxylate; and R1, R2, and R3 are hydrogen;with the provisos that (a) when R1 is sulfate, then R2 is other than H;(b) when R2 is sulfate then R1 is other than H; and R5 is selected fromoptionally substituted alkyl and optionally substituted alkenyl; and nis 0-100.
 71. The method of claim 70, wherein the growth factor is anerve growth factor or fibroblast growth factor.
 72. A method forscreening for a modulator of neuronal growth, wherein the methodcomprises: contacting a cultured neuron with a candidate modulator ofthe following formula:

or a pharmaceutically acceptable derivative thereof, wherein each of R1,R2, R3, and R4 are independently selected as follows: (i) R1 and R2 areeach independently selected from sulfate, phosphate and carboxylate; andR3 and R4 are hydrogen; (ii) R3 and R4 are each independently selectedfrom sulfate, phosphate, carboxylate; and R1 and R2 are hydrogen; (iii)R1, R2 and R3 are each independently selected from sulfate, phosphate,carboxylate; and R4 is hydrogen; (iv) R1, R2, and R4 are eachindependently selected from sulfate, phosphate, carboxylate; and R3 ishydrogen; (v) R2 and R4 are each independently selected from sulfate,phosphate, carboxylate; and R1 and R3 are hydrogen; (vi) R1 is selectedfrom sulfate, phosphate, carboxylate; and R2, R3, and R4 are hydrogen;(vii) R2 is selected from sulfate, phosphate, carboxylate; and R1, R3,and R4 are hydrogen; (viii) R3 is selected from sulfate, phosphate,carboxylate; and R1, R2, and R4 are hydrogen; or (ix) R4 is selectedfrom sulfate, phosphate, carboxylate; and R1, R2, and R3 are hydrogen;with the provisos that (a) when R1 is sulfate, then R2 is other than H;(b) when R2 is sulfate then R1 is other than H; and R5 is selected fromoptionally substituted alkyl and optionally substituted alkenyl; and nis 0-100; (b) determining a percent increase in mean axon length of thecontacted neuron and a percent increase in mean axon length of acorresponding untreated neuron; and (c) comparing the percent increasein mean axon length of the contacted neuron to the percent increase inmean axon length of the corresponding untreated neuron; wherein themodulator yields a difference in percent increase in mean axon length ofthe contacted neuron as compared to the untreated neuron.
 73. The methodof claim 72, wherein the increase in mean axon length of the treatedneuron relative to the untreated neuron is between about 10% to about50%, when the modulator is present in the culture.
 74. The method ofclaim 72, wherein the increase in mean axon length of the untreatedneuron relative to the treated neuron is between about 10% to about 50%,when the modulator is present in the culture.